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Microbes and Infection 14 (2012) 573e583

www.elsevier.com/locate/micinf

Review

Streptococcus pneumoniae: the evolution of antimicrobial resistance


to beta-lactams, fluoroquinolones and macrolides
J.E. Cornick a,b,*, S.D. Bentley c
a

Malawi-Liverpool-Wellcome Clinical Research Programme, PO Box 30096, Chichiri, Blantyre 3, Malawi


b
Institute of Infection and Global Health, The University of Liverpool, Liverpool, UK
c
Wellcome Trust Sanger Institute, Cambridge, UK
Received 11 November 2011; accepted 24 January 2012
Available online 4 February 2012

Abstract
Multi drug resistant Streptococcus pneumoniae constitute a major public health concern worldwide. In this review we discuss how the
transformable nature of the pneumococcus, in parallel with antimicrobial induced stress, contributes to the evolution of antimicrobial resistance;
and how the introduction of the pneumococcal conjugate vaccine has affected the situation.
2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Streptococcus pneumoniae; Evolution; Antimicrobial resistance; Beta-lactams; Fluoroquinolones; Macrolides

1. Introduction
Streptococcus pneumoniae (the pneumococcus) causes
a spectrum of diseases ranging from the relatively mild otitis
media to the life threatening meningitis, pneumonia and
septicemia. Invasive pneumococcal disease (IPD) is defined by
the isolation of pneumococci from normally sterile sites, i.e.
pleural fluid, blood and cerebrospinal fluid. Colonization of
the nasopharynx by S. pneumoniae is a prerequisite for IPD
and also an important step in the development of antimicrobial
resistance because it provides the environment for genetic
recombination and the exchange of genetic material between
pneumococci and other commensal streptococci [1]. In
developed countries half of children are colonized at least
once before they reach their first birthday, while in developing
countries the pneumococcal carriage rate is much higher [2].
Despite the availability of appropriate antimicrobial chemotherapy, the burden of pneumococcal disease in developing

* Corresponding author. Malawi-Liverpool-Wellcome Trust Clinical


Research Programme, PO Box 30096, Chichiri, Blantyre 3, Malawi. Tel.:
265 0 1875918; fax: 265 0 1875774.
E-mail addresses: J.Cornick@liverpool.ac.uk, j.cornick@liv.ac.uk (J.E.
Cornick).

countries has changed very little over the last century [3].
Pneumococcal disease is responsible for 1.6 million deaths
annually, one million of which are children under five in the
developing world [4]. The introduction of penicillin in 1940 led
to a rapid reduction in the morbidity and mortality associated
with pneumococcal disease, however, widespread consumption
and over prescription contributed to the emergence of penicillin
resistance in the 1960s [5]. Today antimicrobial resistant pneumococci constitute a major public health concern; this is in large
part due to the dissemination of multi drug resistant (MDR)
clones [6]. The introduction of the heptavalent pneumococcal
conjugate vaccine (PCV7) was heralded as an important strategy
in the reduction of antimicrobial resistance. The vaccine targets
the cells polysaccharide capsule the structure of which, for each
strain, takes one of 92 different forms and it is this variation
which is reflected in the traditional serotyping scheme; the PCV7
vaccine includes the seven polysaccharides most frequently
associated with disease and more recent vaccines have increased
the valency. In the USA, PCV7 has successfully reduced the
incidence of IPD and antimicrobial resistance in vaccine serotypes; however this has been paralleled by an increase in the
incidence of IPD caused by non-vaccine serotypes amongst
which antimicrobial resistance in increasing, particularly serotype 19A [7]. The introduction of conjugate vaccines with

1286-4579/$ - see front matter 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.micinf.2012.01.012

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J.E. Cornick, S.D. Bentley / Microbes and Infection 14 (2012) 573e583

increased valancy, specifically PCV13, has the potential to


profoundly impact antimicrobial resistance rates amongst
pneumococci.
2. A brief history of antimicrobial resistance
Traditionally infections caused by S. pneumoniae have been
treated with penicillin; the pathogen was highly susceptible
following its introduction in the 1940s. Extensive overuse of
the antimicrobial led to the first report of a penicillin resistant
S. pneumoniae in 1969; a serotype 4 carriage strain isolated
from the throat of a three-year-old boy in New Guinea. The
isolate had a minimum inhibitory concentration (MIC) for
penicillin of 0.5 mg/ml but remained susceptible to chloramphenicol, erythromycin and tetracycline [5]. Little regard was
given to antimicrobial resistance in pneumococci again until
1977 when the attention of the medical community was drawn
to reports of an IPD epidemic in Durban and Johannesburg,
South Africa caused by highly penicillin resistant S. pneumoniae (MIC 2e8 mg/ml). The epidemic strains exhibited
resistance to chloramphenicol, macrolides and tetracycline in
addition to greatly increased MICs to penicillin [8]. Following
this report, MDR pneumococci (defined as strains resistant to
three or more classes of antimicrobials) were reported with
greater frequency worldwide [9].
3. The dominance of multi drug resistance clones
Since the late 1990s molecular typing techniques in tandem
with surveillance studies revealed the worldwide escalation in
MDR strains could largely be attributed to the spread of
a small number of MDR clones. In response to this the
Pneumococcal Molecular Epidemiology Network (PMEN)
was established in 1997 with the aim of standardizing the
nomenclature and classification of pneumococcal clones
worldwide. The nomenclature is defined as the country of
origin, followed by the serotype in superscript and the
consecutive number, (e.g. Spain23F-1 or South Africa19A-7)
[9]. At present PMEN has documented 43 international clones,
26 of which are MDR. The first reported examples of which
was Spain23F-1 or PMEN1. Typically identified as serotype
23F, multi locus sequence type (MLST) 81, this clone is
resistant to multiple antimicrobials including penicillin. It was
first characterized in Spain in the 1980s and subsequently
spread globally. By the late 1990s PMEN1 was estimated to
cause 40% of all penicillin resistant pneumococcal disease in
the USA [10].
Clonal analyses of large collections of pneumococci have
revealed the remarkable dominance that a small number of
clones have amongst the antimicrobial resistant population.
Analysis of 1647 clinical pneumococci strains collected in the
USA (2004e2005) found that 63% were MDR. The majority
(52%) of MDR strains identified in the study were related to
one of two clones, the international clone Taiwan19Fe14
(expressing serotype 19A) or Utah35Be24 [6]. Analysis of
Japanese clinical pneumococcal strains found that ten international clones accounted for 77.8% of MDR strains [11],

while analysis of 151 penicillin non-susceptible (PNSP; this


describes pneumococci that exhibit resistance or intermediate
resistance based on interpretive breakpoints) invasive clinical
strains collected in Poland 2003e2005, found that the vast
majority (90.7%) were MDR clones. The MDR clone Spain9V3 was responsible for 47.5% of PNSP invasive infections,
a significant overrepresentation in the population [12].
4. Driving forces behind the evolution of antimicrobial
resistance
4.1. Widespread use of antimicrobials
Widespread use of antimicrobials is a major driving force
behind the evolution of antimicrobial resistance. Analysis of
31,000 pneumococcal strains from Finland revealed a strong
correlation between macrolide resistance and previous macrolide or azithromycin use. A strong association between
previous beta-lactam or cephalosporin treatment and penicillin
resistance was also found [13]. A study of over 3300 IPD
patients in Canada found that infection with PNSP S. pneumoniae was associated with penicillin, azithromycin or cotrimoxazole treatment in the preceding three months. Patients
who had received macrolides, cotrimoxazole or fluoroquinolones in the three months prior to developing IPD were
four times more likely be infected with pneumococci resistant
to the administered class of antimicrobial than those who had
not been administered antimicrobials [14]. A surveillance
study encompassing 44 research centers and over 1800 clinical
strains in the USA found that macrolide resistance was associated with the use of any class of antimicrobial in the
previous six weeks [15]. The level of antimicrobial
consumption in a geographic region is also an important risk
factor in the development of resistance in that area. Analysis
comparing levels of antimicrobial consumption in 15 European countries during 1998e2004 and 2004e2005 found that
antimicrobial resistance was significantly associated with
the overall rate of antimicrobial use in all of the countries
studied [16].
The strong influence antimicrobial consumption exerts on
the evolution of antimicrobial resistance, is perhaps best
exemplified by the evolution of the MDR clone PMEN2
(Spain6Be2) in Iceland. Penicillin resistant pneumococci were
first detected in Iceland in 1988; subsequently the MDR clone
PMEN2 accounted for 70% of penicillin resistant pneumococcal disease between 1989 and 1992. The clone also
exhibited resistance to cotrimoxazole, chloramphenicol,
tetracycline and erythromycin. PMEN2 remained dominant in
Iceland in the 1990s, however during this time pneumococci
belonging to PMEN2 were reported as exhibiting reduced
MICs to tetracycline and erythromycin. From 1992 to 1996
PMEN2 variants were increasingly isolated from both carriage
and infection that were tetracycline and erythromycin
susceptible. The survival and modest spread of these PMEN2
antimicrobial susceptible variants over a sustained period of
time, strongly suggests that reduced antimicrobial resistance
did not hinder their competitiveness. The emergence of the

J.E. Cornick, S.D. Bentley / Microbes and Infection 14 (2012) 573e583

susceptible variants is hypothesized to be due to a national


campaign urging the Icelandic public and clinicians to show
restraint in their use of antimicrobials; the campaign started in
1992, a short amount of time before the first PMEN2 variant
with reduced antimicrobial resistance was detected [17]. It
should however be noted that a subsequent study found that
despite a reduction in antimicrobial consumption following
the campaign, some MDR PNSP clones increased in frequency
in two of four Icelandic communities studies, highlighting
that other factors influence the selection of antimicrobial
resistance [18].
During or following treatment, subinhibitory concentrations of an antimicrobial can occur within the body. Exposure
to subinhibitory concentrations has been linked to a number of
genotypic and phenotypic changes in pneumococci, including
increase in mutation frequency. The hypermutation phenotype,
characterized by an increase in mutation frequency, results
from permanent mutations to the DNA mismatch repair genes
that normally correct DNA copying errors. During antimicrobial treatment, hypermutation can confer a selective
advantage to pneumococci by allowing them to rapidly accumulate mutations and therefore quickly evolve to cope with
the antimicrobial induced stress conditions. Exposure to
subinhibitory concentrations of ciprofloxacin and streptomycin have been shown to increase mutation rate to rifampicin
resistance in S. pneumoniae between 2 and 5 fold [19].
In addition to promoting hypermutation, antimicrobial
induced stress has been shown to induce transformation in
S. pneumoniae. Competence for genetic transformation is
dependent on the transcriptional activation of the com regulon
which contains the recA gene. The recA protein catalyzes
homologous recombination between internalized DNA and the
recipient pneumococcal genome; it is therefore an essential
component in transformation. Exposure to aminoglycosides
and fluoroquinolone antimicrobials has been shown to activate
transcription of the com regulon, allowing S. pneumoniae to
increase its rate of genetic exchange with co-colonizing
organisms and the possibility of generating a resistant genotype [20]. Clearly this phenomenon will be inherently linked
to the colonization rates of the pneumococcus and related
streptococci implying that the high carriage rates associated
with developing countries would enhance the rates of
dissemination of resistance determinants between strains.
4.2. The impact of PCV on antimicrobial resistance
S. pneumoniae is a Gram-positive, encapsulated diplococcus. The pneumococcal capsular polysaccharide (cps)
protects the bacterium from phagocytosis and is an essential
virulence factor; the unencapsulated laboratory reference
strain R6 is known to be avirulent. To date, 92 serotypes have
been assigned based on differences in the immunochemistry of
the pneumococcal cps. PCV7 contains cps from the seven
serotypes conjugated to non-toxic diphtheria toxoid. Five of
these seven serotypes are strongly associated with antimicrobial resistance; 6B, 9V, 14, 19F and 23F [21]. As discussed
earlier carriage plays an important role in the evolution of

575

antimicrobial resistance as the nasopharynx provides the


environment for recombination between pneumococci and
other commensal streptococci [1]. The reduction in carriage of
vaccine serotypes following the introduction of PCV7 has had
a major impact on antimicrobial resistance incidence amongst
pneumococci.
A longitudinal observational study of IPD in Atlanta,
Georgia showed that following the introduction of PCV7 the
incidence of IPD fell significantly. Coupled with the overall
decrease in IPD was a decrease in IPD caused by antimicrobial
resistant pneumococci. Macrolide resistance in IPD increased
steadily from 4.5/100,000/year in 1994 to 9.3/100,000/year in
1999, however following the introduction of PCV7 in 2001, it
fell to 2.9/100,000/year in 2002 [22]. This is thought to be
largely due to the fact PCV7 encompasses the seven pneumococcal serotypes most commonly associated with antimicrobial resistance. Numerous other studies have documented
a decrease in the incidence of antimicrobial resistance
following the introduction of PCV7. A prospective surveillance study which monitored all pediatric IPD cases admitted
to eight hospitals in the USA over a nine year period found the
incidence of PNSP increased every year 1994e2000, stabilizing at 45% in 2001. After the introduction of PCV7, the
incidence of PNSP fell to 33% in 2002 [23]. Similarly,
surveillance of pediatric IPD cases in Tennessee showed that
the number of IPD cases caused by PNSP fell from 58.8% in
1999 to 30.4% in 2002 after the introduction of PCV7. A
reduction in the number of pediatric IPD cases caused by
cephalosporin and erythromycin resistant pneumococci was
also observed [24].
4.3. Serotype replacement
Data from the Active Bacterial Core surveillance study
shows that the dramatic decrease in the incidence of IPD
caused by vaccine and vaccine related serotypes following the
introduction of PCV7 in 2001 has coincided with a small
increase in resistant IPD caused by non-vaccine serotypes,
especially 19A [7]. A major concern with PCV7 is that out of
92 pneumococcal serotypes it is only effective against the
seven most commonly associated with pediatric infections in
pre-vaccine era USA and other developed countries; this
leaves a large number of serotypes circulating in the population against which the vaccine is not effective. Once
removed from the pneumococcal population, vaccine serotypes no longer play a role in carriage and IPD in the vaccinated population. As a result, an ecological niche is created
which may be taken by non-vaccine serotypes. The succession
of vaccine serotypes by non-vaccine serotypes is termed
serotype replacement and is a pitfall of employing such
a diverse antigen as the capsular polysaccharide in vaccination. Although not anticipated to increase the incidence of
IPD, serotype replacement has the potential to diminish the
positive effects of PCV7.
In the USA 19A strains have been identified as the main
cause of serotype replacement in both carriage and IPD; this
has coincided with a significant increase in penicillin

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resistance and MDR amongst 19A clinical strains. Serotype


19A is now the most frequent cause of drug resistant IPD in
the USA [25]. From 1998 to 2005 the incidence of IPD caused
by 19A increased from 0.8 cases/100,000 population to 2.5
cases/100,000 population, this coincided with an increase of
IPD caused by penicillin resistant 19A from 6.7% to 35%.
Over 70% of penicillin resistant 19A strains belonged to the
rapidly emerging clonal complex 320 (CC320), which is
descended from the MDR Taiwan19F-14, while the remaining
penicillin resistant 19A strains were highly related to CC199.
In 1999, prior to the introduction of PCV7, only CC199 and
three minor clones were apparent among 19A strains. In 2005,
following the introduction of PCV7, 11 clonal sets were
detected including capsular switch variants of serotype 4
clone, ST695 [26]. It should however be noted that not all
replacement serotypes are associated with antimicrobial
resistance; the prevalence of serotype 11A in carriage has
greatly increased following the introduction of PCV7 yet this
serotype shows little association with antimicrobial resistance
[27]. A study monitoring the incidence of IPD in the population of England and Wales before and after vaccine introduction found that there was an increase in the frequency of
IPD caused by non-vaccine serotype following the introduction of PCV7. The major serotypes responsible for replacement were 7F, 19A and 22F. There was however no evidence
for a change in the incidence of resistance to penicillin or
erythromycin in these serotypes following the introduction of
PCV7 [28].
4.4. The introduction of PCV13
PCV7 has played an important role in reducing the burden
of pneumococcal disease in the developing world, however in
some regions, particularly developing countries, PCV7
provides coverage against <50% of invasive disease [29]. To
increase potential coverage against invasive disease, a secondgeneration vaccine, PCV13 has been introduced. PCV13 is
based on the same chemistry as PCV7, but incorporates an
additional six serotypes (1, 3, 5, 6A, 7F and 19A) [30]. Given
that PCV13 is effective against serotype 19A, it has the
potential to reduce the incidence of antimicrobial resistance
caused by the newly emergent CC320. Surveillance studies
have been conducted to assess pneumococcal disease carriage
prior to the introduction of PCV13 [31], this provides the basis
for subsequent studies to investigate if PCV13 successfully
reduces invasive disease caused by antimicrobial resistant
pneumococci, or simply drives the emergence of new serotype
switch variants.
4.5. Transformation and the pneumococcal pan-genome
S. pneumoniae is a naturally competent bacterium, the
genetic plasticity of which was first noted by Avery, Macleod
and McCarty, whose seminal 1944 experiment established
DNA as the transforming particle capable of transferring
instructions for capsule structure and virulence between strains
[32]. S. pneumoniae is capable of incorporating any single

stranded DNA into its genome, therefore recombination can


occur not only between S. pneumoniae but also with related
species that occupy the same ecological niche (i.e. commensal
streptococci). Over the last decade comparative genomics have
given microbiologists an appreciation of how the genomic
plasticity of S. pneumoniae plays a major role in the evolution
of antimicrobial resistance. The transformable nature of S.
pneumoniae has the potential to rapidly introduce novel
phenotypes into a new genetic background and is thus a major
driving force behind the rapid evolution of S. pneumoniae in
response to antimicrobial induced stress conditions [33].
The sum of genomes across bacterial populations can be
described as pan-genomes comprising core and accessory
portions. The core genome consists of genes that are present in
all members of the population and encompass essential functions such as signal transduction and central metabolism. The
accessory genome encodes dispensable genes that are only
present in some member of the species; these genes are not
essential to survival but may confer a fitness advantage in
certain circumstances and include genes encoding antimicrobial resistance mechanisms. Accessory genes are often not
native to S. pneumoniae and have been acquired through
transformation from other bacterial species [34].
The full complement of genes available to individual
strains is present in a pan-genome that is much larger than any
individual genome. Any naturally transformable bacteria that
share the same niche as S. pneumoniae can add to or withdraw
genes from this gene pool [35]. Access to the pan-genome has
resulted in a large degree of genic diversity between pneumococcal clonal complexes; comparative genomics has
revealed that less than half of the pneumococcal pan-genome
is conserved between all strains studied [36]. Phylogenetic
analysis of 44 pneumococcal genomes, encompassing multiple
different serotypes and genetic clones, isolated from both
carriage and invasive disease from a variety of geographic
locations, showed that the majority of pneumococcal strains
belonged to one of six ancient lineages. Distribution of
dispensable genes was consistent with phylogeny but within
a single lineage there was ongoing acquisition and loss of
dispensable genes seen in individual strains. Comparison of
pneumococcal genomes with the genomes of six commensal
streptococci (four Streptococcus mitis, one Streptococcus
infantis and one Streptococcus oralis) revealed access to the
pan-genome is the main driving force behind pneumococcal
evolution. Horizontal gene transfer (HGT) and recombination
between pneumococci and S. mitis was responsible for 30% of
core genome diversity between the latter [37].
The role the pan-genome and genomic plasticity in specific
relation to the evolution of antimicrobial resistance is coming
to light as the number of pneumococcal genomes sequenced
increases. Comparative analysis of the genome of a serotype
14 genome (CGSP14) with four complete genomes and twelve
draft pneumococcal genomes revealed the large contribution
HGT plays in the acquisition of antimicrobial resistance
mechanisms. CGSP14 contains 18 antimicrobial resistance
determinants, nearly half of which are associated with mobile
genetic elements; two conjugate transposons and one genomic

J.E. Cornick, S.D. Bentley / Microbes and Infection 14 (2012) 573e583

island. Variations of the two composite transposons were


found in PMEN1 and S. pneumoniae G54 (serotype 19F),
suggesting that these transposons have experienced frequent
recombination and deletion events in each of the strains,
perhaps as a result of differing selective pressure [38].
Croucher et al. recently adopted a novel genomic approach
to investigate how genetic recombination and point mutations
contribute to the evolution of antimicrobial resistance.
Advanced sequencing technologies enabled the analysis of
complete genome data for a sample of 240 pneumococcal
isolates representing the spread of the PMEN1 clone to 22
countries over a period of 25 years. The analysis showed that
while recombination events occurred at around one tenth of
the frequency of base substitutions they had a much greater
influence on the acquisition of antimicrobial resistance and
vaccine evasion [33].
5. Mechanisms and evolution of antimicrobial resistance
5.1. Beta-lactam resistance
Defining resistance to penicillin is a complex issue. The
Clinical Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing
(EUCAST) regularly publish breakpoints for the interpretation
of antimicrobial susceptibility testing. The breakpoints differ
for meningitis versus other pneumococcal infections and for
parenteral versus oral administration. The term, penicillin nonsusceptible S. pneumoniae (PNSP) refers to pneumococci that
are classed as resistant or intermediately resistant based on
these interpretative breakpoints [39].
The classical mechanism of resistance to penicillin and
other beta-lactams in S. pneumoniae is modification of penicillin binding proteins (PBPs); enzymes involved in the final
step of bacterial cell wall synthesis. The mode of action of
beta-lactams is to bind to PBPS, thereby reducing peptidoglycan synthesis and remodeling. This in turn reduces the
integrity of the bacterial cell wall resulting in inhibited growth
or lysis [40].
Resistance to beta-lactams in pneumococci has arisen from
the development of mosaic pbp genes that show reduced
affinity to the antimicrobial molecule. Six PBPs have been
described in S. pneumoniae, the high molecular weight PBPs;
1a, 1b, 2x, 2a and 2b and the low molecular weight PBP3. The
majority of beta-lactam resistance arises from mutations in
just three of these. Amino acid changes in PBP2b and PBP2x
result in penicillin resistance, while mutations in PBP1a in
tandem with PBP2b and PBP2x increase penicillin MIC
further. PBP2a has also been implicated in penicillin resistance, however it appears to enhance pre-existing resistance
rather than conferring resistance alone [41].
In the vast majority of clinically resistant strains PBPs are
encoded by mosaic genes. Mosaic pbp genes are continuous
nucleotide sequences that differ from non-mosaic alleles by up
to 21%, this level of difference strongly suggests that these
sequence blocks are of non-pneumococcal origin [42]. Resistance mutations are thought to arise through point mutations in

577

pbps in commensal streptococci, blocks of which are then


transformed into S. pneumoniae [41]. Three of the early
recognized penicillin resistant clones (Spain23F-1, Spain6B-2
and Spain9v-3) encode a highly related pbp2x which is thought
to have originated in commensal streptococci. In order to trace
the origin of the conserved mosaic block present in pbp2x
from these Spanish clones, a large collection of oral streptococci from three different continents was analyzed. Sequences
nearly identical to the Spanish mosaic block were present in
an array of pneumococcal clones from countries across all
three continents studied. The size of the mosaic blocks within
the pneumococci and oral streptococci studied were highly
variable, as was the location of recombination sites, implying
that many independent recombination events had contributed
to the blocks dominance. The oral Streptococci that predated
the Spanish clones possessed a larger pbp2x mosaic block.
This strongly suggests that the mosaic block did not originate
from the Spanish clones, but was donated from resistance
commensal Streptococci [43].
Recombination is an important mechanism in the evolution
of beta-lactam resistance, however an increasing body of
evidence suggests that clonal dissemination is also central to
the spread of beta-lactam resistance in pneumococci.
Comparative evolutionary analysis of pbp sequences from 216
clinical pneumococci strains worldwide as part of the Alexander project suggests clonal dissemination plays a more
significant role in the spread of amoxicillin resistance than
HGT events. The study found a high degree of association
between PBP type and ST or clonal complex. The vast
majority (78%) of the amoxicillin resistant strains were
confined within five clonal complexes; HGT played a minimal
contribution to the spread of amoxicillin resistance. HGT of
pbp2a showed the highest frequency, recombinant pbp2a was
found in 10.2% of pneumococci studied, followed by pbp2x
(7.8%) and pbp1a (3.9%) [44].
In addition to mutations in PBPs it appears that a functional
MurMN operon is essential for the expression of beta-lactam
resistance in S. pneumoniae. The MurMN operon encodes
enzymes involved in the branching of muropeptides. Inactivation of MurMN leads to peptidoglycan composed of linear
muropeptides and the virtually complete loss of penicillin
resistance [45]. Analysis of five clinical pneumococcal isolates
with penicillin MICs ranging from 0.25 to 2.0 mg/ml showed that
they all possessed identical PBP and MurMN sequences [46].
While analysis of over 200 clinical isolates from 2005 to 2006
showed a recovery of susceptibility to penicillin despite an
increase in PBP mutations [47]. It therefore appears that in
addition to PBP and the MurMN operon other factors influence
pneumococcal beta-lactam resistance. An example of this is the
two component signal transduction system CiaRH; gene
expression profiling has revealed the CiaRH operon is unregulated in response to subinhibitory concentrations of penicillin
[48]. Research shows CiaRH is necessary for altered pbp2x
genes encoding low affinity PBP2x to be tolerated by S. pneumoniae, even in the absence of antimicrobial stress [49]. Clearly
more investigation is needed to clarify the full role alternative
mechanisms play in the regulation of beta-lactam resistance.

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The acquisition and spread of antimicrobial resistance


within a bacterial population is dependent on a delicate
balance between antimicrobial selective pressure and the cost
resistance places on bacterial fitness. Degree of fitness cost
varies between antimicrobial classes and specific mutations.
The fitness cost imposed by antimicrobial resistance can be
minimized or even eliminated by additional compensatory
mutation. A recent study by Albarracn Orio et al., showed
how pbp2b mutants, in the absence of antimicrobial stress,
were subject to a fitness cost in comparison to wild-type
strains. Acquisition of additional mutations in pbp1a and
pbp2x fully compensated for this fitness cost. Of particular
interest was the finding that these compensatory mutations
also led to increased resistance to beta-lactams. This finding
highlights how antimicrobial resistance can persist in bacterial
populations despite the absence of antimicrobial selective
pressure [50].
Hetero-resistance is another important mechanism proposed
to be employed by pneumococci to reduce any fitness costs
acquired during the evolution of beta-lactam resistance.
Although not yet clearly defined, hetero-resistance is generally
regarded, as the presence of bacterial subpopulations that are
capable of growth in higher antimicrobial concentrations than
the MIC of the whole population would indicate. Heteroresistance has mainly been studied in Staphylococci, but has
now been described in pneumococci. Using population analysis
profiling, Morand and Muhlemann, found evidence of
subpopulations of pneumococci with higher penicillin resistance levels than the majority, in 11 of the 25 strains studied.
Furthermore, seven of the hetero-resistant strains belonged to
MDR clones. It is hypothesized that pneumococci use heteroresistance as a tool to experiment with growth at higher
penicillin MICs without being subjected to the fitness cost
associated with acquisition of resistance mechanisms [51]. To
date beta-lactams are the only group of antimicrobials to which
hetero-resistance in pneumococci has been documented.
5.2. Fluoroquinolone resistance
Originally introduced in the 1980s for the treatment of
gram-negative pathogens, increasing beta-lactam resistance
amongst pneumococci spurred the development of fluoroquinolones effective against gram-positive pathogens.
Although low in relation to beta-lactam resistance, fluoroquinolone resistance is increasing amongst pneumococci,
due in the large to the dissemination of MDR clones [52].
DNA gyrase (GyrA, GyrB) and topoisomerase IV (ParC,
ParE) are vital for DNA supercoiling and chromosome
segregation respectively [53]. Fluoroquinolones bind to target
sides within these proteins, thereby inhibiting pneumococcal
DNA synthesis. Moxifloxacin primarily targets DNA gyrase
(subunit GyrA), while the principal target of ciprofloxacin and
levofloxacin is topoisomerase IV (subunit ParC) [54].
In contrast to beta-lactam resistance, fluoroquinolone
resistance in pneumococci primarily originates from the
alteration of the fluoroquinolone binding site due to the
gradual accumulation of spontaneous mutations in the

quinolone resistance determinant region (QRDR) of gyrA and/


or parC. Biochemical and genetic studies have shown that
mutations altering parC result in resistance to ciprofloxacin,
but do not alone confer resistance to newer fluoroquinolones
[55]. This first step mutation in the QRDR of parC increases
the risk for subsequent mutations that may enhance resistance
further [56]. Most fluoroquinolone resistance 16 mg/ml
originates from mutations in both parC and gyrA. Mutations in
these genes confer resistance to newer fluoroquinolones; levofloxacin, moxifloxacin, gatifloxacin and usually gemifloxacin
[57]. Mutations in gyrB and parE are uncommon and appear
to be inconsequential [58].
The first studies into the genetic basis of fluoroquinolone
resistance in pneumococci found that the most frequent
mutations were in the codons for parC/S79 and gyrA/S81
[59,60]. Analysis of 35 levofloxacin resistant pneumococci,
mostly unrelated, collected worldwide as part of the PROTEKT global surveillance programme found that 51% of
strains carried the same combination of mutations, phenylalanine substitutions at parC/S79 and gyrA/S81 [61]. Other
studies have however suggested the basis of fluoroquinolone
resistance is more heterogeneous. Genetic analysis of the
QRDR region of 14 fluoroquinolone resistant clinical strains,
showed that eight exhibited amino acid mutation at sites other
than parC/S79 and gyrA/S81, these included: gyrA/E474K,
parE/E474K and parC/A63T [62]. In addition to the accumulation of spontaneous mutations, over expression of drug
efflux pumps, PmrA and the ABC efflux pumps PatA and
PatB, have been implicated in the development of fluoroquinolone resistance. Although efflux pumps alone are not
known to confer fluoroquinolone resistance 16 mg/ml, it is
thought that they increase the probability of the acquisition of
additional mutations that confer thus higher level of resistance,
i.e. mutations in parC/E and gyrA/B [63].
In contrast to beta-lactam resistance, the role recombination
plays in the evolution of fluoroquinolone is uncertain. Intraand inter-species transfer of fluoroquinolone resistance loci
has been found to occur in vivo but the frequency of such
events is thought to be rare. Analysis of the QRDR from 71
ciprofloxacin resistant clinical pneumococcal strains, showed
that only one strains possessed a QRDR homologous to viridans groups streptococci, suggesting the impact of recombination on fluoroquinolone resistance is minimal [64]. While
analysis of the QRDR of 46 ciprofloxacin resistant clinical
pneumococcal strains, revealed that five of the strains
exhibited mosaic structures in at least one QRDR. Furthermore, the intergenic parEeparC region in the strains
was larger than is typical in S. pneumoniae and also encoded
an ant gene; the ant gene is present in this region in viridans
group streptococci, strongly suggesting the mosaic QRDR
resulted from recombination with viridans group streptococci.
Again however, these recombination events appear to be
infrequent [65].
From these studies it is clear that viridans group streptococci are acting as a reservoir of fluoroquinolone resistance for
S. pneumoniae. For reasons unknown, viridans group streptococci exhibit higher levels of fluoroquinolone resistance than

J.E. Cornick, S.D. Bentley / Microbes and Infection 14 (2012) 573e583

pneumococci. In vitro studies have reported that inter-species


recombination of fluoroquinolone resistance loci to be 106
times greater than that of spontaneous mutations, however this
level of recombination does not appear to be replicated in vivo
[66]. It is unclear why exchange of resistance loci to viridans
group streptococci and pneumococci does not occur at
a higher frequency. Reduction in fitness cost could be the
explanation, however it has been shown that inter-species
recombination of fluoroquinolone resistance loci does not
have a discernable fitness cost on pneumococci and may even
compensate for some fluoroquinolone resistance mutations
[65].
5.3. Clonal spread of fluoroquinolone resistance
The role of clonal spread in the evolution of fluoroquinolone
resistance is controversial, with studies placing different
weighting on its importance. Analysis of levofloxacin resistant
pneumococci collected by the Active bacterial core surveillance
program at the CDC, USA 1998e2002, found 58% were related
to five international clones, the most common of which was
Spain23F-1, which accounted for 16% of all strains [58]. Analysis
of pneumococcal strains sent to a Spanish reference lab during
2002 found five international clones (Spain23F-1, Spain6B-1,
Spain9V-1, Spain14-5 and Sweden15A-25) accounted for 46.7% of
ciprofloxacin resistant strains [67]. In contrast to this, several
studies have reported that clonal dissemination does not play
a significant contribution in the increase of fluoroquinolone
resistance [61,68,69]. Analysis of levofloxacin resistant pneumococci collected from 25 countries as part of the PROTEKT
study 1999e2000 showed the majority were genetically unrelated, although 34% belonged to the Spain23F-1 clone [61]. These
studies suggest that both clonal dissemination and the emergence
of newly resistant strains contribute to the spread of fluoroquinolone resistance.
5.4. Macrolide resistance
The worldwide increase in penicillin resistance coincided
with an increase in macrolide resistant pneumococci. In many
parts of the world macrolide resistance now exceeds penicillin.
PROTEKT data (1993e2003) shows a global increase in
macrolide resistance, from 31% in 1999 to 36.3% in 2003
[70]. A recent trend is the escalation of macrolide resistance in
countries typically associated with low rates of resistance due
to the dissemination of antimicrobial resistance clones [71].
Macrolides are microbiostatic agents that inhibit bacterial
protein synthesis by binding to the 23S ribosomal RNA. There
are two main mechanisms of macrolide resistance in S.
pneumoniae: modification of the target site or efflux of the
antimicrobial from the cell, through acquisition of the erm or
mef genes respectively.
5.5. erm mediated resistance
Acquisition of the erm(B) gene encoding a 23S RNA
methylase is a major resistant determinant in pneumococci.

579

Expression of the gene results in the post transcriptional


modification of 23S, reducing the affinity of the macrolide to
the 23S binding site. The majority of pneumococci that encode
erm(B) exhibit the MLSB phenotype; resistance to 14-, 15and 16- member ring macrolides, lincosamides and streptogramin B. High level macrolide resistance (>64 mg/ml) is
generally associated with mutations in erm(B) [72]. More
recently a novel erm gene, erm(TR) has also been shown to
confer MLSB resistance [73].
In pneumococci, erm(B) is carried on members of the
Tn916 family of transposons. A number of Tn916 derivatives
carrying erm(B) have been described in pneumococci
(Tn3872, Tn6002, Tn6003, and Tn1545), these transposons
also encode the tet(M) gene conferring tetracycline resistance,
as a result there is a high incidence of tetracycline resistance
amongst macrolide resistant pneumococci. It should however
be noted that the tet(M) gene is not always expressed [74].
One of the earliest derivatives of Tn916 described was
Tn3872, a 23.3k composite element resulting from the insertion of the erm(B) carrying element, Tn917 into Tn916 [75].
The 20.9 kb Tn6002 is the result of the insertion of a 2.8 kb
DNA fragment encoding erm(B) into Tn916 [76]. Tn6003,
a 25.1 kb transposon, is the result of the insertion of the 4.4 kb
macrolide-aminoglycoside-streptothricin (MAS) element into
Tn6002. The MAS element contains aphA-3 and an additional
erm(B), as a result Tn6003 encodes two erm(B) genes [74].
The first derivative of Tn916 described was Tn1545; a 25.3 kb
transposon detected in S. pneumoniae BM4200 [77]. Recent
characterization has shown that Tn1545 is actually 1 kb larger
than originally reported, the transposon results from the
insertion of the 1200 bp putative insertion sequence, IS1239
into Tn6003. In addition to macrolide and tetracycline resistance, Tn1545 and Tn6003 confer resistance to kanamycin and
structurally related aminoglycosides through the presence of
the aphA-3 gene on the MAS element [78].
The macrolide resistance determinant erm(TR), a subclass
of erm(A) was first identified in Streptococcus pyogenes [73].
In 2001 the first documented erm(TR) carrying pneumococcal
strain was isolated from two Greek patients [79]. In contrast to
erm(B), erm(TR) carriage appears to be rare in pneumococci.
Analysis of over 1000 pneumococcal strains from 25 countries
found erm(TR) was present in only 2 strains, both from
Australia [80]. Given the high frequency at which erm(TR) is
present in S. pyogenes, erm(TR) could be expected to circulate
more readily to pneumococci in the nasopharynx [81]. characterization of the erythromycin resistant, S. pneumoniae
AP200 revealed erm(TR) was carried on a relatively large
w56.0 kb genetic element designated, Tn1806. It is possible
that the size of Tn1806 means that it is difficult to acquire and
maintain in the species [82].
There is extensive evidence documenting the dissemination
of erm mediated resistance by clonal spread. Analysis of 109
Spanish erythromycin resistant pneumococci exhibiting the
MLSB phenotype found that five clones (Sweden15A-25, clone19F-ST276, Spain23F-1, Spain23F-2 and clone19A-ST276)
account for over 50% of all MLSB strains. Furthermore, 77.1%
of MLSB strains possess genes relating to Tn916, suggesting

580

J.E. Cornick, S.D. Bentley / Microbes and Infection 14 (2012) 573e583

that macrolide resistance is being disseminated through the


clonal spread of Tn916 family transposons [83].
5.6. mef mediated resistance
Acquisition of mef genes, encoding an active (proton
dependent) efflux pump is the second major mechanism of
macrolide resistance. The majority of mef positive pneumococci exhibit the M phenotype, characterized by resistance to
14- and 15- member ring macrolides. The M phenotypes
exhibit lower erythromycin MICs than the MLSB phenotype,
1e32 mg/ml in comparison to gt; 32 mg/ml [84]. There are at
least three subclasses of mef; the abundant mef(A) and mef(E),
which share 90% sequence identity and the rare variant mef(I)
which has only been described in two Italian clinical strains
[85]. The incidence of mef(A) and mef(E) varies greatly from
country to country, mef(A) dominates in Europe, while mef(E)
dominates in the United States, Asia and South Africa [86].
In pneumococci the three subclasses of mef are carried on
a number of similar but distinct genetic elements, all of these
elements also encode a msr(D) gene that encodes another
efflux pump conferring macrolide resistance. The mef(A) gene
is carried on Tn1207.1, a 7.2 kb defective transposon that
corresponds to the left end of the S. pyogenes transposon
Tn1207.3 [87]. Tn12071.1 is only capable of inserting at one
specific site within the pneumococcal chromosome, clonal
spread is therefore the dominant mechanism of dissemination
of this transposon within the pneumococcal population; the
vast majority of pneumococci carrying Tn1207.1 belong to the
MDR clone, England14-9 [88].
The mef(E) gene is carried on the 5.4e5.5 kb macrolide
efflux genetic assembly (mega) element, similar in sequence to
Tn1207.1 but lacking the region upstream of mef [89]. In
contrast to the mef(A) carrying Tn1207.1, the mega element
has been found inserted at numerous different chromosomal
sites in different serotypes and clonal groups. A novel
composite element, designated Tn2009, resulting from the
insertion of the mega element into a Tn916 like transposon
carrying tet(M) has been also described [90].
A recent trend in macrolide resistance is the emergence of
clinical pneumococcal strains expressing both erm(B) and
mef(E), referred to as the dual phenotype. This trend is the
result of the dissemination of Tn916 derivatives carrying both
genes. Analysis of strains belonging to CC271, a Taiwan19F-14
type strain displaying the dual phenotype, revealed erm(B)
and mef(E) were contained on an novel composite element,
designated Tn2010. Tn2010 genetically similar to Tn2009, but
with the addition of an erm(B) containing element [91].
Another novel composite element has been described in
a Hungarian pneumococcal isolate; sequence type 43,
belonging to CC271. The isolate harbored a new combination
of Tn916, Tn917 and mega has been designated Tn2017 [92].
The novel composite genetic element harboring mef(I) has
recently been characterized and is designated the 5216IQ
complex. The 5216IQ complex consists of two segments; the
first segment is w15.3 kb and is formed from parts of known
transposons, Tn5252 and Tn916. The second segment is

different from any other known genetic element carrying mef.


Designated the IQ element, it ends with identical transposase
genes at both sides and contains the mef(I) gene with an
adjacent new msr(D) gene variant and catQ, a chloroamphenicol acetyltransfrease gene. In comparison to the
elements carrying mef (E), IQ appears to be non mobile,
explaining its scarcity amongst macrolide resistant pneumococci [93].
5.7. The emergence of the dual phenotype
The presence of the dual phenotype is increasingly being
described in macrolide resistant pneumococci worldwide; in
Spain w60%, while in Korea and Russia 30% of macrolide
resistant pneumococci possess both erm(B) and mef(A)
[94e96]. In South Africa 15% of macrolide resistant strains
exhibit the dual phenotype [71]. Data from PROTEKT USA
shows that although the level of macrolide resistance has
stabilized at w29%, the level of resistant pneumococci
exhibiting the dual phenotype has increased from 9.7% in
2000 to 16.4% in 2003. Pneumococci carrying both erm(B)
and mef(A) exhibit a high level of resistance to macrolides in
addition to other antimicrobials. In the USA, 99.2% of macrolide resistant strains exhibit MDR, 90% of these MDR
strains were clonally related to the MDR international Taiwan19F-14 CC271. Data form the PROTEKT global study
(1999e2003) revealed that 83.3% of macrolide resistant
strains exhibiting the dual phenotype were related to CC271
[97]. The high level of resistance teamed with the evidence of
clonal spread by pneumococci possessing both erm(B) and
mef(A), makes the dual phenotype a potentially serious
potential public health problem.
6. Conclusions
Antimicrobial resistant pneumococci have become a major
public health concern worldwide over the past three decades;
due in large to the dissemination of MDR clones. The emergence and maintenance of antimicrobial resistance amongst
the pneumococcal population is reliant on the complex interplay between a number of factors. A high level of antimicrobial consumption in a specific geographic region is perhaps the
biggest driving force behind the emergence of antimicrobial
resistance. Exposure to subinhibitory antimicrobial concentrations can select for a hypermutation phenotype, leading to
the rapid accumulation of resistance mutations in pneumococci. In addition, exposure to antimicrobial stress can induce
competence, promoting the genetic exchange of resistance
mutations between pneumococci and other streptococci
inhabiting the nasopharynx. Whether these newly acquired
resistance mechanisms persist and disseminate within the
pneumococcal population is dependent on the fitness cost they
impose.
In recent years, an additional factor has impacted upon the
evolution of pneumococcal resistance. PCV7 has led to
a reduction in antimicrobial resistance amongst vaccine serotypes, however this has been paralleled by an increase in

J.E. Cornick, S.D. Bentley / Microbes and Infection 14 (2012) 573e583

antimicrobial resistance amongst non-vaccine serotypes. The


introduction of PCV13, encompassing a greater number of
serotypes, has the potential to reduce this problem.
In conclusion, it is clear the development and dissemination
of antimicrobial resistance in the pneumococcal population is
multifaceted; in order to introduce measures to limit the spread
of antimicrobial resistance and prevent the emergence of
resistance to new classes of antimicrobials it is essential that
we gain a better understanding of its evolution.

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