Evolving Resistance Among Gram-positive Pathogens
Evolving Resistance Among Gram-positive Pathogens
Jose M. Munita 2 3 4 6
Arnold S. Bayer 0 2 7
Cesar A. Arias ( 1 2 3 5 6
0 Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center , Torrance
1 Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston
2 Houston , 6431 Fannin St, MSB 2.112, Houston, TX 77030
3 Division of Infectious Diseases, Department of Internal Medicine
4 Clinica Alemana de Santiago, Universidad del Desarrollo , Chile
5 Molecular Genetics and Antimicrobial Resistance Unit, Universidad El Bosque , Bogota , Colombia
6 International Center for Microbial Genomics
7 David Geffen School of Medicine at UCLA , Los Angeles, California
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.
antimicrobial resistance; multidrug-resistant; methicillin-resistant; vancomycin-resistant; penicillin-resistant
that failure to tackle it could jeopardize modern medical
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 . 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 .
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 . 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-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
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 . 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
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) .
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 . Interestingly, the
E447K substitution had been previously selected in vitro by
ceftobiprole passage .
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 . 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 . 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 . In addition, studies on
β-lactams (penicillins with
or without cefazolina)
vraRS, yycFG, graRS, rpoB
vanA Gene cluster
23S rRNA genes, L3/L4
ribosomal proteins, cfr
PBPs (pbp2x, pbp2b in
murM, cpoA, pdgA
23S rRNA genes, L3/L4
ribosomal proteins, cfr
Table 1. Resistance Mechanisms in Clinically Important Gram-positive Pathogens
van Gene clusters
β-lactams (penicillin and
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.
sequence variation of the pbp5 allele identified specific
differlow over the years and has been described in only a few E.
faeences between ampicillin-susceptible and ampicillin-resistant
cium isolates .
isolates (harboring pbp5-S and pbp5-R alleles, respectively),
suggesting evolutionary adaptation . 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,
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
penicilscribed in enterococcal isolates. Initially found in E. faecalis in
lin establish that isolates not causing meningitis are categorized
the early 1980s, the frequency of this mechanism has remained
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
Acquired from enterococci; rare isolates described
Mutations in 23S rRNA genes are main mediators of
resistance; cfr gene is the only transferable determinant
Mutations in pbp2a associated with high-level ceftaroline
resistance (MIC >32 µg/mL)
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
Rare, found mostly in E. faecalis
Several gene clusters described, most acquired on mobile
elements; intrinsic low-level resistance seen in
E. gallinarium and E. casseliflavus (VanC phenotype)
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
Most frequent mutations found in daptomycin-resistant
clinical isolates; many genes involved; resistance
pathways differ between E. faecalis and E. faecium
PBP changes are the most important determinant of
β-lactam resistance; mosaic PBPs are often found in
Rare Rapid development of daptomycin resistance in certain species of streptococci
and ≥8 μg/mL, respectively . In contrast, meningitis isolates
are considered penicillin susceptible (MIC ≤0.06 μg/mL) or
resistant (≥0.12 μg/mL) . The first significant outbreak of
penicillin-resistant S. pneumoniae was reported in the 1970s
and, since then, their frequency has steadily increased
worldwide . Prevalences of β–lactam-resistant pneumococci
vary widely within different regions and are largely shaped by
the use patterns of conjugated pneumococcal vaccines .
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 . 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 .
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
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
. 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 .
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 , 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) . 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 .
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) . Outbreaks caused by
hVISA/VISA strains have also been reported, but their occurrence
continues to be sporadic . 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 . 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 . 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 . 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 .
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
, dalbavancin  and oritavancin . 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 .
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 .
Two oxazolidinones are currently available for the treatment of
gram-positive infections, namely linezolid and tedizolid. Both
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) . 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 .
Since then, it has been found in several bacterial species.
Overall, cfr carriage continues to be sporadic, however, areas of
endemicity have been reported . 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 . 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 .
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
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 . 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 . In
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 .
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) .
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% . 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 . Furthermore, only 3 E.
faecium isolates harbored changes in the L3/L4 ribosomal proteins.
Although cfr has been reported in clinical enterococcal isolates
, its overall frequency continues to be low.
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
. In a 2014 report, no resistance was found in 6691
pneumococci, 2463 viridans streptococci, or 4153 β-hemolytic
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
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  (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 .
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 . 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) . In
addition, DAP-R isolates seem to exhibit important changes in
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 . 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 .
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 .
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 . 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 .
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) . 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 . 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 .
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)
. 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)  and
on the genetic pathway leading to the DAP-R phenotype .
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
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
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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