Resistance to Macrolide Antibiotics in Public
Health Pathogens
Corey Fyfe, Trudy H. Grossman, Kathy Kerstein, and Joyce Sutcliffe
Tetraphase Pharmaceuticals, Watertown, Massachusetts 02472
Correspondence: jsutcliffe@tphase.com
Macrolide resistance mechanisms can be target-based with a change in a 23S ribosomal
RNA (rRNA) residue or a mutation in ribosomal protein L4 or L22 affecting the ribosome’s
interaction with the antibiotic. Alternatively, mono- or dimethylation of A2058 in domain
V of the 23S rRNA by an acquired rRNA methyltransferase, the product of an erm (erythro-
mycin ribosome methylation) gene, can interfere with antibiotic binding. Acquired genes
encoding efflux pumps, most predominantly mef(A) þ msr(D) in pneumococci/streptococci
and msr(A/B) in staphylococci, also mediate resistance. Drug-inactivating mechanisms
include phosphorylation of the 2
0
-hydroxyl of the amino sugar found at position C5 by
phosphotransferases and hydrolysis of the macrocyclic lactone by esterases. These acquired
genes are regulated by either translation or transcription attenuation, largely because cells
are less fit when these genes, especially the rRNA methyltransferases, are highly induced or
constitutively expressed. The induction of gene expression is cleverly tied to the mechanism
of action of macrolides, relying on antibiotic-bound ribosomes stalled at specific sequences
of nascent polypeptides to promote transcription or translation of downstream sequences.
M
acrolide antibiotics are polyketides com-
posed of a 14-, 15-, or 16-membered mac-
rocyclic lactone ring (14-, 15-, and 16-mem-
bered) to which several sugars and/or side
chains have been attached by the producing
organism or as modifications during semisyn-
thesis in the laboratory (Figs. 1 and 2). Newer
semisynthetic derivations, like ketolides teli-
thromycin, and solithromycin, have a C3-keto
group in place of the C3 cladinose (akin to nat-
urally occurring pikromycin) (Brockmann and
Henkel 1950) and an 11,12-cyclic carbamate
with an extended alkyl –aryl side chain that in-
creases the affinity of the antibiotic for the ri-
bosome by 10- to 100-fold (Hansen et al. 1999;
Dunkle et al. 2010); in the case of solithromycin,
a fluorine substituent at C2 provides an addi-
tional ribosomal interaction (Llano-Sotelo et al.
2010). Macrolides continue to be impor tant in
the therapeutic treatment of community-ac-
quired pneumonia (Streptococcus pneumoniae,
Haemophilus influenzae, Moraxella catarrhalis,
and atypicals Legionella pneumophila, Myco-
plasma pneumoniae, Chlamydia pneumoniae),
sexually transmitted diseases (Neiserria gonor-
hoeae, Chlamydia trachomatis, Mycoplasma gen-
italium), shigellosis, and salmonellosis. With
solithromycin heading for a new drug applica-
tion (NDA) filing in 2016 and having the in
vitro potency to treat erythromycin-resistant
Editors: Lynn L. Silver and Karen Bush
Additional Perspectives on Antibiotics and Antibiotic Resistance available at www.perspectivesinmedicine.org
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pneumococci and gonococci (Farrell et al.
2015; Hook et al. 2015), macrolides/ketolides
will continue as an impor tant part of the anti-
biotic armamentarium.
The mechanism of action of macrolides has
been further refined through a combination of
genetic, biochemical, crystallographic, and ri-
bosome profiling studies (Tu et al. 2005; Dunkle
et al. 2010; Kannan et al. 2012, 2014; Gupta et
al. 2016). Macrolides/ketolides are sensed by
the ribosome and, in the presence of certain
macrolide-stalling nascent amino acid chain-
dependent motifs, selectively inhibit protein
synthesis. Further, and to different extents, ke-
tolides and macrolides cause frameshifting,
leading to aberrant protein synthesis.
Shortly after its clinical debut in 1953, resis-
tance to erythromycin in staphylococci was de-
scribed and was likely mediated by methylation
of the 23S ribosomal RNA (rRNA) at nucleotide
A2058 (Escherichia coli numbering) encoded
by an erythromycin ribosomal methyltransfer-
ase (erm) gene (Weisblum 1995a). Erm methyl-
transferases add one or two methyl groups
to the N-6 exocyclic amino group of A2058,
disrupting the key hydrogen bond between
A2058 and the desosamine sugar at C5 (Fig.
3). Ribosomal methylation by methyltransfer-
ases encoded by erm genes remains the most
widespread macrolide resistance in pathogenic
bacteria, with certain erm genes more predom-
inantly found in some species. Streptococci
CH
3
CH
3
CH
3
N(CH
3
)
2
CH
3
CH
3
CH
3
H
3
C
HO
R
3
OCH
3
CH
3
R
1
9
R
2
C
2
H
5
O
O
O
O
O
OH
OH
1
4
O
CH
3
OH
O
R
1
R
2
R
3
Erythromycin
Clarithromycin
Roxithromycin
O
NOCH
2
OC
2
H
4
OCH
3
OOH
OH
OCH
3
O
R
1
R
F
H
Solithromycin
Telithromycin
NH
2
N
N
N
N
N
N
CH
3
CH
3
H
3
C
R
2
CH
3
N(CH
3
)
2
CH
3
OH
CH
3
H
3
C
H
3
C
HO
OCH
3
H
3
C
R
1
N
C
2
H
5
O
OH
O
O
O
HO
4
O
O
OH
R
1
CH
3
N(CH
3
)
2
CH
3
H
3
C
CH
3
OCH
3
H
3
C
H
3
C
O
N
O
H
3
C
O
R
C
2
H
5
O
O
O
O
O
HO
1
Oleandomycin H
COCH
3
Troleandomycin
R
1
, R
2
, R
3
Azithromycin
Tulathromycin CH
2
NH(CH
2
)
2
CH
3
CH
3
H
H
R
2
R
1
CH
3
OCH
3
OR
3
CH
3
CH
3
CH
3
N(CH
3
)
2
O
O
H
3
C
H
3
C
H
3
C
C
2
H
5
O
OR
1
O
O
O
R
2
O
O
O
Figure 1. Structures of 14- and 15-membered macrolides.
C. Fyfe et al.
2
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generally have erm(B) or erm(A), subclass
erm(TR), whereas erm(A), erm(B), or erm(C)
are found in staphylococci and erm(F) in an-
aerobes and H. influenzae (Table 2, and refer-
ences therein; see also faculty.washington.edu/
marilynr). There can be either mono- or di-
methylation of A2058 and the degree of rRNA
dimethylation can determine ketolide resistance
(Douthwaite et al. 2005). Most erm genes are
inducible by 14- and 15-membered macrolides,
whereby translation repression of the erm meth-
yltransferase gene, because of the sequestration
of its ribosome-binding site (RBS) by messen-
ger RNA (mRNA) secondary structure, is re-
lieved by binding of the inducer to the ribosome
(Horinouchi and Weisblum 1980; Depardieu
et al. 2007; Subramaniam et al. 2011). Upstream
of the start codon of a methyltransferase gene is
an open reading frame (ORF) that produces
leader peptides of different lengths (8 –38 ami-
no acids), each containing a macrolide stalling
motif; when the macrolide-bound ribosome
pauses, the attenuator, a stem and loop struc-
ture that encompasses the RBS, is disrupted,
resulting in ribosomal binding and synthesis
of the methyltransferase (Subramaniam et al.
2011; Arenz et al. 2014a,b). Although most
erm genes are regulated by translation attenua-
tion, a few genes (e.g., erm(K)) are regulated by
transcription attenuation (Kwak et al. 1991;
Choi et al. 1997) or through inducible tran-
scription factors (Morris et al. 2005). Ketolide
induction has been described for erm(C) and
involves promotion of frameshifting in the
erm(C) leader (ermCL ) mRNA, leading to by-
pass of the ermCL stop codon, via rearrange-
ment of the secondary mRNA structure, allow-
ing expression of the downstream resistance
gene (Gupta et al. 2013a).
There are two families of macrolide efflux
pumps with regulation that is at least in part,
transcriptionally mediated—mef, a major-facil-
O
O
O
O
O
O
O
N
O
O-R
2
O-R
3
O-R
4
O-R
1
H
3
C
Spiramycin
Josamycin
Kitasamycin
Midecamycin
Rosamicin
Tilmicosin
Tylosin
Propionyl Propionyl
CH
3
COCH
3
COCH
2
CH(CH
3
)
2
Forosamine
R
1
R
2
R
3
R
4
HH
H
H
H
H
H
H
H
H
H
3
C
O
O
OH
O
O
O
O
O
N
O
O
O
O
O
O
O
O
O
O
O
OH
R
1
H
3
C
H
3
C
R
1
Desosamine
Mycaminose-mycarose
Figure 2. Structures of 16-membered macrolides.
Resistance to Macrolide Antibiotics in Public Health Pathogens
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C2610
U2609
U2609
C2610
A752
ERY
G2505
Desosamine
Desosamine
C2611
A2059
A2058
C2611
C2
C2
90°
F
F
G2057
A752
A2058
A752
A2059
Alkyl–aryl
group
Alkyl–aryl
group
C2611
A2058
A752
G2505
A
C
B
A2059
C2610
C2611
Cladinose
U2609
A2058
A2059
Desosamine
G2505
23S rRNA
ERY
TEL
SOL
SOL
23S rRNA
23S rRNA
Figure 3. A model based on the crystal structure of the 70S Escherichia coli ribosome bound to erythromycin
(PDB ID codes 3OFO, 3OFP, 3OFR, 3OFQ), telithromycin (PDB ID codes 3OAQ, 3OAR, 3OAS, 3OAT), and
solithromycin (PDB ID code 4WWW) (Dunkle et al. 2010; Llano-Sotelo et al. 2010). (A) A comparison of the
conformations of erythromycin (ERY, magenta), telithromycin (TEL, gold), and solithromycin (SOL, green) in
their binding sites at the top of the nascent peptide exit tunnel (PET) comprised of 23S ribosomal RNA (rRNA).
23S rRNA residues are marked, with nitrogen in dark blue and oxygen in red. Hydrogen bonds are indicated
between residues by dotted lines, including between residues U2609 in domain V and A752 in domain II of 23S
rRNA. The alkyl –aryl arm of telithromycin and solithromycin is shown stacking with A752. (B) Erythromycin-
only view. The key hydrogen bond between the 2
0
hydroxyl of the desosamine and the N1 of A2058 is indicated.
The exocyclic N6 amino group that is methylated by Erm methyltransferases is notable next to the N1 of A2058.
(C) Solithromycin-only view. The left side of the figure displays solithromycin in the same conformation as
macrolides in A and B. The C2-F is visible through the ring of C2611, but a better view of its interaction with
C2611 is displayed when the view is rotated by 90
˚
, with the C2-F stacking with the hydrophobic side of C2611.
C2611 is paired through three hydrogen bonds to G2057.
C. Fyfe et al.
4
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itator-superfamily pump that confers resistance
to most 14- and 15-membered macrolides (Le-
clercq and Courvalin 2002; Sutcliffe and Leclercq
2002; Chancey et al. 2011) and msr, a member
of the ATP-binding cassette (ABC) superfamily
that generally confers resistance to 14- and 15-
membered macrolides and streptogramin B
and low-level resistance to ketolides (Sutcliffe
and Leclercq 2002; Chancey et al. 2011).
Intrinsic efflux pumps that are not specific
to macrolides exist in different species. These
pumps are often responsible for limiting macro-
lide spectrum in Gram-negative species and
overexpression of multidrug efflux pumps is as-
sociated with clinically relevant drug resistance
in both Gram-negative and Gram-positive spe-
cies. Interested readers are referred to recent
reviews (Costa et al. 2013; Blair et al. 2014; Del-
mar et al. 2014; Sun et al. 2014).
Mutations in 23S rRNA, L4, and/or L22 ri-
bosomal proteins can confer macrolide resis-
tance because the mutation is technically in the
23S rRNA gene. In addition, macrolides can be
inactivated by esterases or phosphotransferases
(in public health pathogens and macrolide
producers) or by glycosyltransferases (decribed
in many strains of Streptomyces producing poly-
ketides or polyether antibiotics; Micromonospora
purpurea; Nocardia asteroides), deacylases (N.
asteroides), or formyl reductases (N. asteroides,
Nocardia brasiliensis, Nocardia otitidisca viarum)
(Sutcliffe and Leclercq 2002; Roberts 2008;
Shakya and Wright 2010; Morar et al. 2012).
Many strains carry more than one macrolide
resistance mechanism, sometimes on the same
mobile element.
This review will focus on antimicrobial re-
sistance mechanisms to macrolides primarily in
public health pathogens. Recent reviews on the
mechanisms of macrolide resistance are recom-
mended (Leclercq and Courvalin 2002; Sutcliffe
and Leclercq 2002; Franceschi et al. 2004; De-
pardieu et al. 2007; Roberts 2008; Kannan and
Mankin 2011; Wilson 2014) as well as the web-
site for macrolide–lincosamide–streptogramin
resistances maintained by Marilyn Roberts (see
faculty.washington.edu/marilynr). Based on the
paper published in 1999 that set out to prevent
duplicate genes being renamed when discovered
in a new species or as part of a novel mobile
element (Roberts et al. 1999), macrolide-resis-
tant genes are considered in the same family if
they have 80% amino acid identity from the
original gene identified for that family.
MACROLIDE MECHANISM OF ACTION
All members of the macrolide class inhibit bac-
terial protein synthesis by binding to the 23S
rRNA in the large ribosomal subunit (50S)
downstream from the peptidyltransferase center
(PTC), the catalytic site for peptide bond for-
mation (for overview of protein synthesis, see
Arenz and Wilson 2016) (Wilson 2009, 2014;
Dunkle et al. 2010; Kannan et al. 2014). Macro-
lides/ketolides bind at the entrance of the pep-
tide exit tunnel (PET) just above the constric-
tion formed by extended loops of ribosomal
proteins L4 and L22 (Yusupov et al. 2001; Da-
vydova et al. 2002; Hansen et al. 2002; Schlun-
zen et al. 2003; Tu et al. 2005; Dunkle et al.
2010), further restricting the effective diameter
of the PET. The macrocyclic lactone and the C5
sugars overlap (Fig. 3A). The sugar at C5 (often
desosamine) is positioned toward the PTC, and
macrolides like tylosin that have a disaccharide
at the C5 position, reach deeper into the PTC.
The 2
0
hydroxyl of desosamine sugar at C5
makes a key hydrogen bond contact w ith the
N1 atom of A2058 and modification at this po-
sition by either mutation or methylation of the
N6 exocyclic amine results in macrolide resis-
tance (see Fig. 3B) (Sutcliffe and Leclercq 2002;
Franceschi et al. 2004; Tu et al. 2005; Dunkle
et al. 2010). Other residues help define a local
binding conformation for macrolides, includ-
ing G2057 and C2611 that form a Watson–
Crick base pair with each other and to which
the hydrophobic face of the lactone ring is
packed (seen best in Fig. 3C). For ketolides, te-
lithromycin, and solithromycin, the extended
alkyl– aryl arm of each drug is oriented down
the tunnel and makes a stacking interaction
with a base pair formed by A752 and U2609
in the 23S rRNA (Fig. 3A,C); these side chains
align closely in the crystal structure of each drug
complexed to E. coli 70S ribosome, but are po-
sitioned differently from the crystal structures
Resistance to Macrolide Antibiotics in Public Health Pathogens
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