SUPPLEMENT ARTICLE
Evolving Resistance Among Gram-positive
Pathogens
Jose M. Munita,1,5,7 Arnold S. Bayer,3,4 and Cesar A. Arias1,2,5,6
1
Division of Infectious Diseases, Department of Internal Medicine, and 2Department of Microbiology and Molecular Genetics, University of Texas Medical
School at Houston; 3Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, and 4David Geffen School of Medicine at UCLA,
Los Angeles, California; 5International Center for Microbial Genomics, and 6Molecular Genetics and Antimicrobial Resistance Unit, Universidad El Bosque,
Bogota, Colombia; and 7Clinica Alemana de Santiago, Universidad del Desarrollo, Chile
Antimicrobial therapy is a key component of modern medical practice and a cornerstone for the development of
complex clinical interventions in critically ill patients. Unfortunately, the increasing problem of antimicrobial
resistance is now recognized as a major public health threat jeopardizing the care of thousands of patients
worldwide. Gram-positive pathogens exhibit an immense genetic repertoire to adapt and develop resistance
to virtually all antimicrobials clinically available. As more molecules become available to treat resistant
gram-positive infections, resistance emerges as an evolutionary response. Thus, antimicrobial resistance has
to be envisaged as an evolving phenomenon that demands constant surveillance and continuous efforts to identify emerging mechanisms of resistance to optimize the use of antibiotics and create strategies to circumvent this
problem. Here, we will provide a broad perspective on the clinical aspects of antibiotic resistance in relevant
gram-positive pathogens with emphasis on the mechanistic strategies used by these organisms to avoid being
killed by commonly used antimicrobial agents.
Keywords. antimicrobial resistance; multidrug-resistant; methicillin-resistant; vancomycin-resistant;
penicillin-resistant.
The discovery and commercialization of antimicrobials
revolutionized modern medicine and became a major
tool for the development of complex medical interventions, such as cancer therapy, organ transplantation,
and management of the critically ill. Unfortunately, antimicrobial resistance is now threatening the care of
such patients. Indeed, infections caused by multidrugresistant (MDR) organisms are associated with significant mortality and carry an important economic
burden, estimated at >$20 billion per year, just in the
United States [1]. Furthermore, the World Health Organization has identified antimicrobial resistance as one of
the most serious health threats worldwide and suggested
Correspondence: Cesar A. Arias, MD, PhD, University of Texas Medical School at
Houston, 6431 Fannin St, MSB 2.112, Houston, TX 77030 ([email protected].
edu).
Clinical Infectious Diseases® 2015;61(S2):S48–57
© The Author 2015. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
[email protected].
DOI: 10.1093/cid/civ523
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that failure to tackle it could jeopardize modern medical
achievements [2].
MDR gram-positive organisms are major human
pathogens, causing both healthcare- and communityassociated infections. Among them, methicillin-resistant
Staphylococcus aureus (MRSA), vancomycin-resistant
Enterococcus faecium (VRE), and drug-resistant Streptococcus pneumoniae have been designated as serious
public threats by the US Centers for Disease Control
and Prevention [1]. Indeed, MRSA and VRE are leading causes of healthcare-associated infections in the
United States, with conservative estimates suggesting
they cause >12 000 deaths per year. Similarly, infections
due to drug-resistant S. pneumoniae, the main cause of
bacterial pneumonia and meningitis in adults, are estimated to cause 19 000 excess admissions and 7000
deaths per year in the United States alone [1].
The genetic and biochemical bases of antimicrobial
resistance in gram-positive bacteria are diverse and
they frequently differ within genera and/or species.
Further, evolution of bacterial resistance is tightly
influenced by the presence of environmental stressors, among
which the use (and misuse) of antimicrobials is thought to
play a major role. For instance, exposure of S. pneumoniae to
antibiotics has been shown to activate the transcription of a
gene regulon that results in increased genetic exchange with
co-colonizing organisms and the subsequent development of
resistant strains [3]. In this article, we will focus on the mechanisms of resistance in clinically important gram-positive bacteria, with emphasis on the most relevant antimicrobials used to
manage severe infections caused by these organisms.
β-LACTAM RESISTANCE
β-lactams disrupt cell-wall synthesis by mostly inhibiting
transpeptidase activity of penicillin-binding proteins (PBPs)
that cross-link the nascent peptidoglycan. Penicillin resistance
was described shortly after penicillin discovery, prompting the
search for novel and more potent β-lactams. However, the discovery of each new β-lactam compound has been consistently
followed by emergence of resistance, highlighting that antimicrobial pressure is a major driving force of bacterial evolution.
There are 2 main mechanisms of β-lactam resistance in grampositive pathogens, enzymatic degradation through the production of β-lactamases and decreased affinity of the antibiotic for
its target, usually either by acquisition of exogenous DNA encoding a low-affinity PBP or through changes in the native pbp genes
(Table 1).
β-Lactam Resistance in S. aureus
Emergence and dissemination of β-lactam resistance in S. aureus occurred in several epidemic waves that could be summarized by (1) acquisition of a plasmid-encoded penicillinase in
the 1950s ( particularly the clone known as phage type 80/81),
(2) a second wave that started after the introduction of methicillin (1959) and was characterized by the emergence of hospital-associated MRSA isolates that mainly circulated in European
hospitals until the 1970s, (3) the development of several highly
successful hospital-associated MRSA clones that spread in
different parts of the world, and continue to circulate (third
wave), and (4) the last wave of MRSA resistance to date, characterized by the emergence and dissemination of communityassociated MRSA in the 1990s [4]. Nowadays, most S. aureus
are penicillin-resistant through the production of a plasmidborne β-lactamase (4 types are known, including A, B, C, and D).
Although the anti-staphylococcal penicillins (eg, nafcillin)
are stable against these enzymes, staphylococcal-type A β-lactamase
has higher rates of cefazolin hydrolysis, and some methicillinsusceptible S. aureus (MSSA) isolates harboring this enzyme exhibit the so-called cefazolin inoculum effect. This phenomenon
involves increased minimum inhibitory concentrations (MICs)
to cefazolin when susceptibility testing is performed using a higher
inoculum (107 colony-forming units/mL) compared with the
standard inoculum (105 colony-forming units/mL). Although
the clinical relevance of this phenomenon is still controversial,
some evidence suggests that this phenotype may have important
clinical implications in high-inocula infections, such as infective
endocarditis [5–7].
The mechanism of methicillin resistance relies on acquisition of
the staphylococcal chromosomal cassette mec (SCCmec) containing mecA, which encodes PBP2a, a transpeptidase with low affinity for all β-lactams (except for last-generation cephalosporins).
Expression of mecA is tightly regulated and it often requires induction by a β-lactam. There are a number of different variants
of the SCCmec DNA; however, in general, shorter elements that
lack resistance determinants to other antimicrobials are present in
community-associated MRSA (SCCmec IV or V) [8].
Ceftaroline and ceftobiprole are new additions to the β-lactam
family with potent activity against MRSA because of their enhanced
affinity for PBP2a. Ceftaroline resistance (MIC ≥4 μg/mL) in
clinical MRSA is uncommon to date, but reports of isolates with
MICs between 4 and 8 μg/mL have been published [9–11], although the mechanisms leading to this phenotype are unclear.
During 2014, an MRSA isolate with a high ceftaroline MIC
(>32 μg/mL; ST5) was recovered from the bloodstream of a patient previously treated with ceftaroline in Houston, Texas. The
mechanism of resistance was associated with 2 contiguous substitutions (Y446N and E447K) in the penicillin-binding pocket
of the transpeptidase domain of PBP2a [12]. Interestingly, the
E447K substitution had been previously selected in vitro by ceftobiprole passage [13].
β-Lactam Resistance in Enterococci
Treatment of enterococcal infections has long been considered a
therapeutic challenge. Indeed, the MICs of penicillin and ampicillin (the most active anti-enterococcal β-lactams) are much
higher than for other common gram-positive organisms, probably owing to the expression of lower affinity PBPs (eg, PBP5),
and enterococcal isolates are often tolerant to β-lactam antibiotics (ie, lack of bactericidal activity in vitro), complicating the
treatment of serious infections [14]. Furthermore, high-level
resistance to ampicillin (MICs ≥64 μg/mL) is a hallmark of
healthcare-associated MDR isolates of E. faecium. Conversely,
ampicillin resistance continues to be strikingly uncommon in
Enterococcus faecalis isolates.
The mechanisms of high-level ampicillin resistance in enterococci are not completely understood but they have been
most consistently associated with changes in the pbp5 gene, encoding a PBP with low affinity for β-lactams [15]. A variety of
mutations have been described and although the individual
contribution to resistance of each genetic change is unclear, some
of them (eg, M485A plus insertion of Ser466) have been directly
linked to the resistance phenotype [16]. In addition, studies on
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Table 1. Resistance Mechanisms in Clinically Important Gram-positive Pathogens
Pathogen and
Resistance Strategy
Staphylococci
Drug inactivation
Antibiotic
Gene(s) Involved
Comments
Plasmid-encoded β-lactamase that hydrolyzes penicillins;
isoxazolyl penicillins (eg, nafcillin), cephalosporins,a and
carbapenems are stable
PBP2a has low affinity for β-lactams (except ceftaroline and
ceftobiprole)
β-lactams (penicillins with
or without cefazolina)
blaZ
Target replacement
β-lactams
mecA
Target modification
Vancomycin (VRSA)
Linezolid
vanA Gene cluster
23S rRNA genes, L3/L4
ribosomal proteins
cfr
Acquired from enterococci; rare isolates described
Mutations in 23S rRNA genes are main mediators of
resistance; cfr gene is the only transferable determinant
of LNZ-R
Ceftaroline
pbp2a
Mutations in pbp2a associated with high-level ceftaroline
resistance (MIC >32 µg/mL)
Vancomycin (VISA)
vraRS, yycFG, graRS, rpoB
Daptomycin
mprF, dlt,
vraRS, yycFG, pgsA, cls
Many genes involved in the resistance phenotype, most
related to regulatory systems
Many genes involved in the resistant phenotype; main
pathway seems to be repulsion of the antibiotic through
an increase in the net positive charge of the cell envelope
Changes in cell
surface
adaptation
Enterococci
Drug inactivation
β-lactams (penicillins)
blaZ
Rare, found mostly in E. faecalis
Target replacement
Vancomycin
van Gene clusters
Several gene clusters described, most acquired on mobile
elements; intrinsic low-level resistance seen in
E. gallinarium and E. casseliflavus (VanC phenotype)
Target modification
β-lactams (penicillin and
ampicillin)
pbp5 b
Linezolid
23S rRNA genes, L3/L4
ribosomal proteins, cfr
PBP5-R differs from PBP5-S in only about 5% of amino acid
sequence; certain amino acid substitutions have been
directly implicated in ampicillin resistance (see text)
Rare, mutations in 23S rRNA and L3/L4 are similar to those
found in staphylococci, as is the cfr gene
Daptomycin
liaFSR, yycGF, gdpD,
cls
Most frequent mutations found in daptomycin-resistant
clinical isolates; many genes involved; resistance
pathways differ between E. faecalis and E. faecium
β-lactams (penicillin)
PBPs (pbp2x, pbp2b in
pneumococci),
murM, cpoA, pdgA
PBP changes are the most important determinant of
β-lactam resistance; mosaic PBPs are often found in
penicillin-resistant isolates
Linezolid
23S rRNA genes, L3/L4
ribosomal proteins, cfr
Rare
Daptomycin
Unknown
Rapid development of daptomycin resistance in certain
species of streptococci
Changes in cell
surface
adaptation
Streptococci
Target modification
Unknown
Abbreviations: LNZ-R, linezolid resistance; MIC, minimum inhibitory concentration; PBP, penicillin-binding protein; rRNA, ribosomal RNA; VISA, vancomycinintermediate Staphylococcus aureus; VRSA, vancomycin-resistant S. aureus.
a
The ability of certain types of staphylococcal β-lactamase to hydrolyze cefazolin may cause therapeutic failures.
b
PBP5 (encoded by pbp5) is part of the core genome of enterococci. Because of its decreased affinity for penicillin, it is responsible for the natural tolerance of these
organisms to β-lactams.
sequence variation of the pbp5 allele identified specific differences between ampicillin-susceptible and ampicillin-resistant
isolates (harboring pbp5-S and pbp5-R alleles, respectively),
suggesting evolutionary adaptation [17]. Indeed, PBP5-R and
PBP5-S sequences differ in approximately 5%, at the amino
acid level, although no particular change has been consistently
correlated with specific increases in the ampicillin MIC. Finally,
β-lactamase–mediated ampicillin resistance has also been described in enterococcal isolates. Initially found in E. faecalis in
the early 1980s, the frequency of this mechanism has remained
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low over the years and has been described in only a few E. faecium isolates [18].
β-Lactam Resistance in Pneumococci
Streptococcus pneumoniae is naturally susceptible to most antimicrobials, including penicillins, long considered the first-line
therapy for pneumococcal infections. Current Clinical and Laboratory Standards Institute break points for parenteral penicillin establish that isolates not causing meningitis are categorized
as susceptible, intermediate, or resistant, with MICs of ≤2, 4,
and ≥8 μg/mL, respectively [19]. In contrast, meningitis isolates
are considered penicillin susceptible (MIC ≤0.06 μg/mL) or resistant (≥0.12 μg/mL) [19]. The first significant outbreak of
penicillin-resistant S. pneumoniae was reported in the 1970s
and, since then, their frequency has steadily increased worldwide [20]. Prevalences of β–lactam-resistant pneumococci
vary widely within different regions and are largely shaped by
the use patterns of conjugated pneumococcal vaccines [21].
The main mechanism of pneumococcal β-lactam resistance is
through changes in the native PBPs by recombination with exogenous pbp genes, in a process that relies on the pneumococcal
ability to incorporate naked DNA from the environment (transformation). Point mutations in pbp genes also play a role and
may act synergistically to further increase the MICs. Recombinant PBPs of resistant isolates are highly variable and classically
show mosaic blocks that derive from the recombination with
external pbp genes (mostly from Streptococcus mitis and Streptococcus oralis) [22, 23]. Strikingly, the magnitude of the recombination can be such that it may change the serotype of the
isolate. Mosaicism in PBP2x and PBP2b is the most frequent
change associated with β-lactam resistance. Moreover, mutational changes in PBP1a can result in high-level resistance
when they occur in the background of low-affinity mosaic
PBP2x or PBP2b [24]. Alterations in the remaining PBPs have
been occasionally related to resistance, but their frequency is
lower. Finally, mutations in other genes, such as murM (involved in the biosynthesis of the murein), pdgA (encoding a
GlcNAc deacetylase), and cpoA (that codes for a glycosyltransferase) have also been uncommonly associated with β-lactam
resistance in pneumococci [24].
GLYCOPEPTIDE RESISTANCE
Glycopeptides prevent cross-linking of peptidoglycan by binding to the terminal D-alanine-D-alanine (D-Ala-D-Ala) of
peptidoglycan precursors, inhibiting cell-wall synthesis. Vancomycin was first approved for clinical use in 1958, and for many
years has been the “workhorse” antibiotic for the treatment of
MRSA infections. Despite its heavy use, it took >40 years for the
first clinical isolates with high-level resistance to vancomycin to
emerge [25].
Vancomycin-Resistant Enterococci
VRE isolates were first described as community commensals in
1986 in Europe, and were associated with the use of avoparcin, a
glycopeptide used in animal husbandry. Consequently, avoparcin use was banned, and the frequency of VRE decreased. Later,
VRE emerged in the United States (where avoparcin was never
approved), mainly in clinical isolates recovered from the hospital environment. Since then, VRE prevalence in the United
States has steadily increased, becoming one of the most
challenging causes of healthcare-associated infections. Importantly, there are considerable interspecies differences, with
most vancomycin resistance occurring in E. faecium isolates
[26]. The successful spread of E. faecium has been tracked to
the dissemination of a hospital-associated genetic clade that is
often MDR, also harboring ampicillin and high-level aminoglycoside resistance determinants [27].
The mechanism of VRE is acquisition of van gene clusters,
probably from environmental organisms. The most frequent
gene cluster is vanA, usually located in a Tn3-family transposon
(Tn1546) that has been found in conjugative and nonconjugative plasmids. In addition, 8 other van clusters (vanBCDEGLMN) have been characterized in enterococcal isolates.
These gene clusters encode a complex enzymatic machinery
that modifies the terminal D-Ala-D-Ala termini of peptidoglycan precursors and destroys the “normal” D-Ala-D-Ala ending
precursors. Most frequently, D-Ala-D-Ala is replaced for
D-Ala-D-lactate, decreasing the affinity of vancomycin for its
target by approximately 1000-fold. D-Ala-D-Ala can also be
replaced for D-Ala-D-serine, which affects the affinity of vancomycin for its target to a lesser degree (resulting in relatively lowlevel resistance, with MICs of 4–32 μg/mL).
In general, the van clusters harbor 3 classes of genes: those
encoding a 2-component regulatory system that tightly controls
the expression of the resistance genes, those involved in the
biochemical pathway for synthesis of D-lactate or D-serine,
and those involved in the destruction of normal D-Ala-DAla-ending peptidoglycan precursors, encoding proteins with
dipeptidase and carboxypeptidase activities. The biochemical
pathways of vancomycin resistance in enterococci have been extensively described [28], and a detailed explanation is beyond
the scope of this review.
Vancomycin Resistance in Staphylococci
Reduced vancomycin susceptibility in staphylococci can be categorized by 2 distinct phenotypes: (1) most commonly, isolates
with intermediate susceptibility to vancomycin (vancomycinintermediate S. aureus [VISA]; MIC, 4–8 μg/mL), and (2) rarely, high-level resistance to vancomycin (vancomycin-resistant
S. aureus [VRSA]; MIC ≥16 μg/mL) [29]. The first VISA isolate
(Mu50), reported in 1997, was derived from a vancomycinsusceptible strain (Mu3), later found to have a small subpopulation
with MICs >2 μg/mL, a condition now designated heterogeneous
VISA or hetero-VISA (hVISA). The hVISA/VISA phenotype
usually arises in vivo in patients with invasive staphylococcal infections who received a prolonged course of vancomycin [30].
Thus, although the overall prevalence of hVISA/VISA is low, it
can reach up to 30% in selected clinical scenarios using refined
assays to detect resistant subpopulations (eg, patients with
MRSA and infective endocarditis) [31]. Outbreaks caused by
hVISA/VISA strains have also been reported, but their occurrence
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continues to be sporadic [32]. Interestingly, the VISA phenotype has also been described in a minority of MSSA strains.
Although the genetic basis of hVISA/VISA seems to be complex
and not completely understood, the available evidence suggests
that it requires a number of ordered and sequential mutations
that usually involve several regulatory systems controlling cellenvelope homeostasis [33]. Among them, the genes most consistently implicated are walkR (also known as yycFG), vraRS
(homolog of liaFSR), and graRS [34, 35]. Mutations in rpoB
(encoding the RNA polymerase subunit B) have also been linked
with the VISA phenotype. These genetic changes produce important remodeling of the cell envelope, resulting in a thickened cell
wall that seem to harbor increased amounts of free D-Ala-D-Ala
dipeptides with reduced cross-linking [30]. Overall, these perturbations may “trap” vancomycin in outer layers of the peptidoglycan, preventing its ability to reach its target of peptidoglycan
precursors emerging from the cytoplasm.
The first reported case of VRSA occurred in Michigan in
2002 and resulted from the acquisition of the vanA gene cluster
from a vancomycin-resistant E. faecalis [36]. However, in contrast to VRE, VRSA remain rare, with only 13 isolates described
in the United States and a total of 4 additional cases reported
from Brazil, Portugal, Iran, and India [33, 37]. Most VRSA
strains have been recovered as “colonizers” from the skin of diabetic patients; 12 of the 13 cases found in the United States
correspond to MRSA belonging to CC5, the most common
clonal complex associated with MRSA hospital dissemination
in the country (the remaining isolate belongs to CC30). Importantly, a Brazilian MRSA isolate carrying the vanA gene cluster
(ST8) was found to be related to a community-associated MRSA
genetic lineage with high dissemination capacity in Latin America (USA300, Latin American variant). Most worrisome, the
vanA gene cluster in this isolate was found in a highly transferable staphylococcal plasmid that was also acquired by a different
bloodstream isolate of MSSA within the same patient [37].
Finally, 3 antibiotics from the lipoglycopeptide family have
been approved by the US Food and Drug Administration for
the treatment of gram-positive infections, namely, telavancin
[38], dalbavancin [39] and oritavancin [40]. All of these compounds appear to be active in vitro against hVISA/VISA isolates.
Of these 3 drugs, oritavancin retains some in vitro activity against
enterococcal strains exhibiting the VanA phenotype [41]. Nonetheless, its clinical efficacy against these latter strains remains to
be established, and initial studies in animal models suggest that
selection of resistant mutants is likely when oritavancin is used
as monotherapy [42].
OXAZOLIDINONE RESISTANCE
Two oxazolidinones are currently available for the treatment of
gram-positive infections, namely linezolid and tedizolid. Both
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compounds inhibit protein synthesis through interactions
with the A site of the 50S ribosomal subunit. Mechanisms of
oxazolidinone resistance include (1) mutations in the genes encoding the 23S ribosomal RNA (rRNA), (2) changes in the L3/
L4 ribosomal proteins, and (3) methylation of the 23S rRNA by
a methylase designated as Cfr (chloramphenicol-florfenicol resistance) (Figure 1). Overall, mutations in the central loop of the
domain V of the 23 rRNA are the most frequent determinants
of linezolid resistance (LNZ-R). Although several mutations
have been described, the most frequent change found in clinical
isolates is G2576T (Escherichia coli numbering). Importantly,
because bacteria carry multiple copies of the 23S rRNA genes,
mutations need to accumulate in several copies to increase the
MIC (gene-dose effect) [43]. Substitutions in the L3 and L4 ribosomal proteins are also associated with development of LNZR in vivo and in vitro, both alone and in combination with other
resistance determinants. Interestingly, mutations in the L3/L4
seem to be particularly frequent in coagulase-negative staphylococci (see below).
Cfr encodes a methyltransferase whose target is position
A2503 of the 23S rRNA. Initially reported in a Staphylococcus
sciuri recovered from cattle, cfr was first described in humans in
2005 in a S. aureus isolated from a patient in Colombia [44].
Since then, it has been found in several bacterial species. Overall, cfr carriage continues to be sporadic, however, areas of endemicity have been reported [45]. Furthermore, cfr-positive
isolates have been associated with outbreaks of LNZ-R S. aureus
and coagulase-negative staphylococci in different clinical settings [46, 47]. Cfr is the only transmissible LNZ-R determinant
and it has been associated with various mobile genetic elements.
In addition, it is frequently cotransmitted with other resistance
determinants that affect protein synthesis, and it seems to carry
a low fitness cost [48]. Altogether, these characteristics suggest
that the potential for dissemination of this MDR trait is high. It
is also important to note that in contrast to mutational resistance, in vivo data suggested that cfr-mediated LNZ-R could
be overcome with higher doses of the antibiotic [49]. Importantly, carriage of cfr does not seem to confer resistance to tedizolid,
the other oxazolidinone approved in 2014 by the Food and
Drug Administration for the treatment of skin and soft-tissue
infections [50].
LNZ-R in Staphylococci
Two comprehensive surveillances collecting worldwide isolates
over a 10-year-period showed that staphylococcal isolates have
remained highly susceptible to linezolid [45]. As mentioned,
changes in L3/L4 ribosomal proteins are particularly frequent
in Staphylococcus epidermidis, with 1 study finding them in 89
of 122 LNZ-R isolates (73%). In terms of frequency, cfr was present in 13% of S. aureus and in 15% of 182 LNZ-R coagulasenegative staphylococci from postmarketing surveillance [45]. In
Figure 1. Schematic representation of the mechanism of action and resistance to linezolid. A, Linezolid interferes with the positioning of aminoacyl
transfer RNA (tRNA) by interactions with the peptidyl transferase center. Ribosomal proteins L3 and L4, associated with resistance, are shown.
B, Representation of domain V of 23S ribosomal RNA (rRNA) showing mutations associated with linezolid resistance. Position A2503, the target of Cfr
(chloramphenicol-florfenicol resistance) methylation, is highlighted. Abbreviation: mRNA, messenger RNA.
addition, several outbreaks of cfr-positive S. aureus and S. epidermidis have been reported, and a 2013 report implicated cfr in
local endemicity of LNZ-R in a hospital in Madrid [51]. Tedizolid resistance has been described in vitro, and the main mechanism relates to changes in the 23S rRNA (including the
presence of a dual-mutation T2571C plus G2576T) [52].
LNZ-R Enterococci
The first described LNZ-R clinical isolates were E. faecium recovered from patients receiving linezolid as part of a compassionate program. As seen in other bacteria, the prevalence of
LNZ-R enterococci has remained low. Data from 6718 enterococcal isolates tested from 2004 to 2012 showed that the prevalence of LNZ-R consistently remained <0.9% [53]. Mutations in
the 23S rRNA genes are the most frequently identified changes.
In fact, of 48 LNZ-R E. faecalis and 121 E. faecium, these
mutations were the only identified resistance trait in 73% and
88% of isolates, respectively [45]. Furthermore, only 3 E. faecium isolates harbored changes in the L3/L4 ribosomal proteins.
Although cfr has been reported in clinical enterococcal isolates
[54], its overall frequency continues to be low.
LNZ-R Streptococci
Very few cases of LNZ-R streptococci have been described, including sporadic reports of LNZ-R S. pneumoniae, Streptococcus sanguinis, and S. oralis. In addition, the cfr gene was
reported from a Streptococcus suis isolate of porcine origin
[55]. In a 2014 report, no resistance was found in 6691 pneumococci, 2463 viridans streptococci, or 4153 β-hemolytic streptococci [53].
DAPTOMYCIN RESISTANCE
Daptomycin (DAP) is a bactericidal lipopeptide antibiotic active against a wide range of gram-positive clinical pathogens.
It damages the cell membrane (CM) in a calcium-dependent
manner, interacting with its target mostly at the bacterial
division septum. Importantly, the presence of specific CM
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phospholipids (eg, phosphatidylglycerol [PG]) is crucial for
the antibacterial action of DAP [56, 57]. Development of DAP
resistance (DAP-R) during therapy seems to be an important
problem affecting the clinical efficacy of DAP. The exact mechanisms of DAP-R remain to be elucidated, but current knowledge suggests that they are complex, diverse, and multifactorial,
arising from mutational changes (Table 1).
DAP-R in S. aureus
Most S. aureus remain DAP susceptible (DAP-S), but reports
documenting the emergence of DAP-R continue to accumulate,
particularly in the context of high-inoculum infections and/or
after exposure to vancomycin [58, 59]. It has been postulated
that the mechanism of DAP-R in S. aureus is mediated by electrostatic repulsion of the DAP calcium complex from the cell
surface, mainly by increasing the positive charge of the bacterial
surface [60] (Figure 2). The most common gene implicated in
the phenotype is mprF, which encodes a bifunctional enzyme
(MprF) that incorporates the positive charged amino acid lysine
to PG. The resulting lysyl-PG, a positively charged phospholipid, is translocated from the inner to the outer leaflet of the CM
by the same enzyme [61].
Several mprF mutations have been associated with DAP-R,
most frequently producing a “gain of function” phenotype.
The increased positive charge of the cell envelope is thought
to impair the binding of DAP to the CM target. The DAP repulsion theory has also been supported by the finding of DAPR strains overexpressing the dlt operon, responsible for the
incorporation of D-alanine ( positively charged amino acid) into
cell-wall teichoic acids [62]. However, repulsion is not the only
explanation for DAP-R staphylococci, because not all isolates
with mprF mutations exhibit significant changes in surface
charge, and DAP-R isolates with either no mutations in mprF
or without gain in function in the enzyme have been described.
Other determinants implicated in DAP-R include genes encoding enzymes involved in phospholipid metabolism, such PG
and cardiolipin synthetases ( pgsA and cls, respectively) [63]. In
addition, DAP-R isolates seem to exhibit important changes in
Figure 2. Proposed mechanisms of action and resistance to daptomycin in Gram-positive organisms. A, The daptomycin-calcium complex interacts with
the cell membrane mainly at septal areas. B, In Enterococcus faecalis, the antibiotic is diverted from the septum (black arrow) in a process associated with
redistribution of anionic phospholipid microdomains (eg, cardiolipin) away from the septal plane. C, In Staphylococcus aureus and Enterococcus faecium, the
positively charged daptomycin-calcium complex is “repelled” from the cell surface in a process associated with increase in the net positive charge of the
cell envelope (not all strains). Abbreviations: DAP-R, daptomycin-resistant; DAP-S, daptomycin-susceptible.
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cell-wall homeostasis, similar to those observed in VISA isolates. The 2-component regulatory systems VraSR and YycFG
(WalKR) are thought to play a particularly important role in development of DAP-R [59]. Indeed, mutations in yycFG have
been directly implicated in DAP-R, and inactivation of vraSR
in a DAP-R isolate caused reversion to DAP-S [64]. Interestingly, 1 study showed that up to 80% of VISA isolates were DAP-R
[59, 65]. In addition, DAP-R emerged in a patient with MRSA
endocarditis in whom vancomycin therapy failed after a VISA
phenotype developed, although never exposed to DAP [35].
DAP-R in Enterococci
Enterococci are less susceptible than staphylococci to DAP
(Clinical and Laboratory Standards Institute break point, 4fold that of S. aureus). Most genes implicated in DAP-R in enterococci seem to be grouped into 2 broad categories: those
encoding regulatory systems that orchestrate cell-envelope homeostasis and stress response and those coding for enzymes
involved in CM phospholipid metabolism. Interestingly, the
mechanisms of DAP-R seem to differ between E. faecium and
E. faecalis (Figure 2).
Genomic analysis of a clinical strain pair of E. faecalis identified 3 genes responsible for DAP-R in vivo [66]. Two of them
(gdpD and cls encoding a phosphodiesterase and cardiolipin
synthase, respectively) were involved in phospholipid metabolism; the remaining locus (liaF) was part of a 3-component regulatory system (LiaFSR, a homologue of the S. aureus VraTSR
system described above) that orchestrates the cell-envelope
stress response in gram-positive organisms. Changes in LiaFSR
are thought to be the initial and pivotal modifications contributing to the development of DAP-R in vivo. Furthermore, deletion of liaR, the response regulator of the LiaFSR system,
resulted in complete reversal of the DAP-R phenotype [67].
Most importantly, the mechanism of resistance does not seem
to be mediated by repulsion of the antibiotic from the cell surface; instead, the lack of killing by DAP has been best correlated
with a “redistribution” of CM cardiolipin microdomains away
from the septum mediated by the LiaFSR system, a change that
seems to prevent the interaction of DAP with its main septal
target (the “diversion” hypothesis; see Figure 2) [68]. In addition, changes in the CM phospholipid composition (mainly decreased PG and increased cardiolipin) seem to be paramount in
DAP-R in E. faecalis.
Recent evidence suggests that the “repulsion” mechanism is
more likely to mediate DAP-R in E. faecium (Figure 2), although the genetic basis of resistance appears to be similar to
that of E. faecalis. Furthermore, changes in LiaFSR were also frequently found in DAP-S clinical E. faecium isolates with MICs
close to the break point (3–4 μg/mL), and their presence was
sufficient to abolish the bactericidal activity of DAP. These
data suggested that such mutations could increase the risk of
therapeutic failure, irrespective of the MIC [69]. Of note, a
2014 report of DAP failure in a patient with VRE bacteremia
caused by a DAP-S isolate (MIC, 3 μg/mL) harboring liaFSR
mutations further supports the role of these genes in the
DAP-R phenotype [70].
DAP-R in Streptococci
Reports of DAP-R streptococcal strains are rare, and large surveillance programs consistently show that streptococcal isolates
remain almost uniformly susceptible to DAP. Importantly, recent evidence suggests that high-level and durable DAP-R can
rapidly arise in vivo and in vitro when viridans group streptococci are exposed to DAP [71, 72]. However, the actual mechanism of this form of DAP-R has not been elucidated.
Development of DAP-R seems to be correlated with an increased susceptibility to β-lactam antibiotics (“see-saw” effect)
[73]. Indeed, several reports have suggested that the DAP-βlactam combination is efficacious and bactericidal against
DAP-R isolates [73–76]. Although the mechanism of this apparent synergy has not been fully elucidated, it seems to be
dependent on specific PBPs (ie, PBP-1 in S. aureus) [77] and
on the genetic pathway leading to the DAP-R phenotype [78].
CONCLUSION
Resistance to front-line antibiotics is an evolving phenomenon
in gram-positive bacterial pathogens. Despite the availability of
new molecules to treat infections caused by these organisms, the
optimal therapeutic approaches remain to be established. The
immense genetic plasticity of the most clinically relevant
gram-positive bacteria poses important challenges for designing
strategies to curtail development of resistance. As new compounds become available, the adaptive bacterial response will
probably emerge in clinical settings. Therefore, efforts to optimize the use of antibiotics with gram-positive spectrum and
identify emerging mechanisms of resistance should be clinical
priorities.
Notes
Financial support. Editorial support, funded by Theravance Biopharma Antibiotics, Inc, was provided by Envision Scientific Solutions. This
work was supported by the Chilean Ministry of Education, Clinical Alemana
de Santiago, and Universidad del Desarrollo School of Medicine, Chile
(grants to J. M. M.); and by the National Institute of Allergy and Infectious
Diseases, National Institutes of Health (grants R01 AI39108–15 to A. S. B.
and R01 AI093749 to C. A. A.).
Supplement sponsorship. This article was published as part of a
supplement titled “Telavancin: Treatment for Gram-positive Infections–
New Perspectives on In Vitro and Clinical Data,” sponsored by Theravance
Biopharma Antibiotics, Inc.
Potential conflicts of interest. The authors received no financial compensation for the preparation of this article. A. S. B has provided expert testimony to Johnson, Graffe, et al; Morrow, Kidman, Tinker, et al; Galloway,
Lucchese, et al; MES Solutions; Hoffman, Sheffield, et al; and Clifford
Gram-positive Resistance
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CID 2015:61 (Suppl 2)
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Law. C. A. A. has received lecture fees, research support, and consulting fees
from Pfizer; lecture and consulting honoraria from Novartis, Cubist, Forest
Pharmaceuticals, AstraZeneca and Bayer Pharmaceuticals; and research
support from Forest Pharmaceuticals, Theravance, and Theravance Biopharma Antibiotics, Inc. J. M. M. reports no potential conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
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