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Antimicrobial Resistance in Neisseria gonorrhoeae in the 21st Century: Past, Evolution, and Future

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Neisseria gonorrhoeae is evolving into a superbug with resistance to previously and currently recommended antimicrobials for treatment of gonorrhea, which is a major public health concern globally. Given the global nature of gonorrhea, the high rate of usage of antimicrobials, suboptimal control and monitoring of antimicrobial resistance (AMR) and treatment failures, slow update of treatment guidelines in most geographical settings, and the extraordinary capacity of the gonococci to develop and retain AMR, it is likely that the global problem of gonococcal AMR will worsen in the foreseeable future and that the severe complications of gonorrhea will emerge as a silent epidemic. By understanding the evolution, emergence, and spread of AMR in N. gonorrhoeae, including its molecular and phenotypic mechanisms, resistance to antimicrobials used clinically can be anticipated, future methods for genetic testing for AMR might permit region-specific and tailor-made antimicrobial therapy, and the design of novel antimicrobials to circumvent the resistance problems can be undertaken more rationally. This review focuses on the history and evolution of gonorrhea treatment regimens and emerging resistance to them, on genetic and phenotypic determinants of gonococcal resistance to previously and currently recommended antimicrobials, including biological costs or benefits; and on crucial actions and future advances necessary to detect and treat resistant gonococcal strains and, ultimately, retain gonorrhea as a treatable infection.
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Antimicrobial Resistance in Neisseria gonorrhoeae in the 21st Century:
Past, Evolution, and Future
Magnus Unemo,
a
William M. Shafer
b,c
WHO Collaborating Centre for Gonorrhoea and Other Sexually Transmitted Infections, National Reference Laboratory for Pathogenic Neisseria, Department of Laboratory
Medicine, Microbiology, O
¨rebro University Hospital, O
¨rebro, Sweden
a
; Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta,
Georgia, USA
b
; Laboratories of Bacterial Pathogenesis, Veterans Affairs Medical Center, Decatur, Georgia, USA
c
SUMMARY ..................................................................................................................................................587
INTRODUCTION ............................................................................................................................................588
GONORRHEA ...............................................................................................................................................588
DIAGNOSIS OF GONORRHEA AND DETECTION OF ANTIMICROBIAL RESISTANCE IN NEISSERIA GONORRHOEAE ......................................589
Diagnosis of Gonorrhea ..................................................................................................................................589
Detection of Antimicrobial Resistance in N. gonorrhoeae.................................................................................................589
HISTORY OF TREATMENT REGIMENS AND RESISTANCE EVOLUTION ....................................................................................590
The Preantimicrobial Era..................................................................................................................................590
The Antimicrobial Era.....................................................................................................................................590
Sulfonamides ..........................................................................................................................................590
Penicillin ...............................................................................................................................................590
Tetracycline ............................................................................................................................................591
Spectinomycin.........................................................................................................................................591
Quinolones ............................................................................................................................................592
Macrolides .............................................................................................................................................592
Cephalosporins ........................................................................................................................................592
MECHANISMS OF ANTIMICROBIAL RESISTANCE IN N. GONORRHOEAE ..................................................................................593
Resistance Emergence and Spread.......................................................................................................................593
Sulfonamide Resistance ..................................................................................................................................593
Penicillin Resistance ......................................................................................................................................593
Plasmid-mediated penicillin resistance ................................................................................................................593
Chromosomally mediated penicillin resistance........................................................................................................595
Tetracycline Resistance ..................................................................................................................................595
Plasmid-mediated tetracycline resistance .............................................................................................................595
Chromosomally mediated tetracycline resistance .....................................................................................................595
Spectinomycin Resistance................................................................................................................................596
Quinolone Resistance ....................................................................................................................................596
Macrolide Resistance .....................................................................................................................................596
Cephalosporin Resistance ................................................................................................................................597
Increased Efflux and Decreased Influx of Antimicrobials .................................................................................................598
Increased efflux ........................................................................................................................................598
Decreased influx .......................................................................................................................................599
ANTIMICROBIAL RESISTANCE AND GONOCOCCAL FITNESS..............................................................................................599
ENVIRONMENTAL CONDITIONS AND GONOCOCCAL RESISTANCE TO ANTIMICROBIALS ...............................................................600
INTERNATIONAL RESPONSES TO RESISTANCE EVOLUTION AND POSSIBLE EMERGENCE OF UNTREATABLE GONORRHEA............................601
FUTURE PERSPECTIVES FOR TREATMENT..................................................................................................................601
ACKNOWLEDGMENTS......................................................................................................................................603
REFERENCES ................................................................................................................................................603
AUTHOR BIOS ..............................................................................................................................................613
SUMMARY
Neisseria gonorrhoeae is evolving into a superbug with resistance
to previously and currently recommended antimicrobials for
treatment of gonorrhea, which is a major public health concern
globally. Given the global nature of gonorrhea, the high rate of
usage of antimicrobials, suboptimal control and monitoring of
antimicrobial resistance (AMR) and treatment failures, slow up-
date of treatment guidelines in most geographical settings, and the
extraordinary capacity of the gonococci to develop and retain
AMR, it is likely that the global problem of gonococcal AMR will
worsen in the foreseeable future and that the severe complications
of gonorrhea will emerge as a silent epidemic. By understanding
the evolution, emergence, and spread of AMR in N. gonorrhoeae,
including its molecular and phenotypic mechanisms, resistance to
antimicrobials used clinically can be anticipated, future methods
for genetic testing for AMR might permit region-specific and tai-
lor-made antimicrobial therapy, and the design of novel antimi-
crobials to circumvent the resistance problems can be undertaken
more rationally. This review focuses on the history and evolution
of gonorrhea treatment regimens and emerging resistance to
Address correspondence to William M. Shafer, wshafer@emory.edu.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/CMR.00010-14
July 2014 Volume 27 Number 3 Clinical Microbiology Reviews p. 587– 613 cmr.asm.org 587
them, on genetic and phenotypic determinants of gonococcal re-
sistance to previously and currently recommended antimicrobi-
als, including biological costs or benefits; and on crucial actions
and future advances necessary to detect and treat resistant gono-
coccal strains and, ultimately, retain gonorrhea as a treatable in-
fection.
INTRODUCTION
The sexually transmitted infection (STI) gonorrhea remains a
significant global public health concern. This requires imme-
diate international attention and resources because the global bur-
den of infection is increasing (1), and Neisseria gonorrhoeae
(gonococcus), the etiological agent of gonorrhea, is evolving into a
superbug and may become untreatable due to its resistance to all
classes of antimicrobials available for treating infections. Gonor-
rhea has been treated successfully by use of antimicrobials for the
past 70 to 80 years. However, internationally, there is now a high
prevalence of N. gonorrhoeae strains with resistance to most anti-
microbials previously and currently widely available for treatment
(e.g., sulfonamides, penicillins, earlier cephalosporins, tetracy-
clines, macrolides, and fluoroquinolones). The recent occurrence
of failures to treat gonorrhea with the extended-spectrum cepha-
losporins (ESCs) cefixime and ceftriaxone and the emergence of
gonococcal strains exhibiting high-level clinical resistance to all
ESCs (2–5), combined with resistance to nearly all other available
therapeutic antimicrobials, have caused great concern, as evi-
denced by publications in the medical literature (5–9) and the lay
press (10) and by development of global, regional, and national
action/response plans (11–14). In most settings worldwide, ceftri-
axone is the last remaining option for empirical first-line antimi-
crobial monotherapy. Due to this fact, there is fear that gonorrhea
might become untreatable using antimicrobial monotherapy. In
response to this concern, recommendations to use dual-antimi-
crobial therapy, i.e., mainly ceftriaxone and azithromycin, have
been introduced in the United States (15), the United Kingdom
(16), and all of Europe (17). Unfortunately, the susceptibility of
gonococcal isolates to ceftriaxone has been decreasing globally,
and resistance to azithromycin is easily selected and already prev-
alent in many settings. Accordingly, these dual-antimicrobial reg-
imens might not be effective long-term solutions and, addition-
ally, are not affordable in many resource-poor settings (5,8).
Furthermore, more expensive antimicrobials, such as high-quality
ceftriaxone, are frequently not available even for monotherapy in
low-resource settings.
The looming public health crisis of antimicrobial-resistant
gonococci cannot be understated, as treatment regimens will most
certainly become more expensive, and as treatment failures occur,
medical costs will increase substantially as a result of severe com-
plications that compromise the general and reproductive health of
infected individuals (11–14). Given the global nature and large
burden of gonorrhea, the high-level and frequently uncontrolled
usage of antimicrobials, suboptimal control and monitoring of
antimicrobial resistance (AMR) and treatment failures, slow up-
date of treatment guidelines in most geographical settings, and the
extraordinary capacity of the gonococci to develop and retain
AMR, the global problem of gonococcal AMR will likely worsen in
the foreseeable future, and the severe complications of gonorrhea
will emerge as a silent epidemic.
By understanding the evolution, emergence, and spread of
AMR in N. gonorrhoeae, including its molecular and phenotypic
mechanisms, resistance to antimicrobials used clinically can be
anticipated, future methods for genetic testing for AMR might
permit region-specific and tailor-made antimicrobial therapy,
and the design of novel antimicrobials to circumvent the problem
of resistance can be undertaken more rationally. This review fo-
cuses on the history and evolution of gonorrhea treatment regi-
mens and emerging resistance to them, on genetic and phenotypic
mechanisms of gonococcal resistance to previously and currently
recommended antimicrobials, including biological costs or bene-
fits, and on crucial actions and advances necessary to detect and
treat resistant gonococcal strains and, ultimately, retain gonor-
rhea as a treatable infection.
GONORRHEA
Gonorrhea (“the clap”; the term was introduced in 1378 and likely
descended from the name of the old Parisian district where pros-
titutes were housed, i.e., Les Clapiers) is an ancient disease with
biblical references (Old Testament; Leviticus 15:1–3). The obli-
gate pathogen N. gonorrhoeae infects only humans in nature and
causes urethritis in men and cervicitis in women. A minority of
men (10%) but a large proportion of women (50%) can have
asymptomatic urogenital infections. Rectal and pharyngeal gon-
orrhea, which is commonly asymptomatic, is mostly identified in
men who have sex with men (MSM); however, depending on sex-
ual practice, it can be found in both sexes. If the urogenital infec-
tion remains undetected or not appropriately treated, it might
ascend to the upper genital tract and result in many severe repro-
ductive complications (especially, but not exclusively, in women),
such as endometritis, pelvic inflammatory disease, penile edema,
and epididymitis, resulting in infertility or involuntary loss of life
through ectopic pregnancy. The failure to curb the transmission
of gonorrhea also promotes the transmission of other STIs, in-
cluding HIV infection (14,17–20). Conjunctivitis can occur in
adults, but most commonly, infection of the eye presents as oph-
thalmia neonatorum in the newborn, which can result in blind-
ness. Disseminated gonococcal infection can occur in both sexes
but is nowadays rarely encountered (14,17,20).
In 2008, the World Health Organization (WHO) estimated
106 million new cases of gonorrhea among adults globally. This
was a 21% increase compared to the number in 2005. The highest
estimates were in the WHO Western Pacific Region (42.0 million
cases), WHO South-East Asia Region (25.4 million cases), and
WHO Africa Region (21.1 million cases) (1). However, the num-
bers of reported cases, especially from low-resource settings, are
substantially smaller. This is due to suboptimal diagnostics (lack
of appropriate methods or access to testing and use of syndromic
management) and/or incomplete case reporting and epidemio-
logical surveillance. These problems can result in substantial, un-
recognized morbidity and hidden health care costs for countries.
Accordingly, gonorrhea, including its severe complications,
causes substantial morbidity and socioeconomic consequences.
Public health control of gonorrhea relies totally on appropriate
antimicrobial treatment, together with generalized and targeted
prevention efforts, use of effective diagnostics, partner notifica-
tion processes, and epidemiological surveillance. Therapy should
cure individual cases to reduce the risk of complications and pre-
vent further transmission of the infection. Considering the large
number of annual estimated cases (106 million) worldwide (1),
AMR in N. gonorrhoeae has considerable implications for control
of gonorrhea, including its severe complications, in communities
Unemo and Shafer
588 cmr.asm.org Clinical Microbiology Reviews
globally. The prospect of gonococcal strains exhibiting multidrug
resistance (MDR) and extensive drug resistance (XDR), including
resistance to ceftriaxone, the last remaining option for first-line
empirical monotherapy in many settings, is cause for great con-
cern. XDR gonococcal strains are those resistant to 2 (MDR
strains are resistant to 1) of the antibiotic classes currently gener-
ally recommended for treatment (ESCs [oral and injectable ones
are considered separately] and spectinomycin) and 3 (MDR
strains are resistant to 2) of the classes now less frequently used
or proposed for use (e.g., penicillins, fluoroquinolones, azithro-
mycin, aminoglycosides, and carbapenems) (19).
DIAGNOSIS OF GONORRHEA AND DETECTION OF
ANTIMICROBIAL RESISTANCE IN NEISSERIA GONORRHOEAE
Diagnosis of Gonorrhea
Guidelines for the diagnosis of gonorrhea have been described
previously (17,20). As aforementioned, gonorrhea is frequently
asymptomatic, and if symptoms are present, they are commonly
nonspecific. Accordingly, appropriate laboratory diagnostics are
crucial for confirmed diagnosis, case finding, and test of cure. The
diagnosis of gonorrhea is established by detection of N. gonor-
rhoeae or its genetic material in genital or extragenital specimens
by microscopy of stained smears, culture, or nucleic acid amplifi-
cation tests (NAATs). Only evaluated and quality-assured meth-
ods should be used. Ideally, AMR testing of gonococcal isolates
should be an integral part of the laboratory diagnosis.
Gonococci can be identified as intracellular diplococci in
polymorphonuclear leukocytes by microscopy (magnification,
1,000) of Gram- or methylene blue-stained smears. This
method is cheap, provides rapid results, and has a high sensitivity
and specificity for the diagnosis of symptomatic men with urethral
discharge. However, microscopy is not recommended as the only
method for diagnosis of cervical, pharyngeal, or rectal gonorrhea,
or for asymptomatic patients, because negative results do not ex-
clude infection, due to the low sensitivity of the method. Further-
more, the performance characteristics highly depend on the expe-
rience of the microscopist. Importantly, this method does not
provide any AMR data.
Culture, the old “gold standard,” offers high sensitivity and up
to 100% specificity (if appropriate species-verifying assays are ap-
plied) and is the only established method that enables complete
AMR testing. Nevertheless, the method is relatively slow, and to
obtain high sensitivity and specificity, it is crucial to strictly opti-
mize the conditions for sample collection, transport, and storage
and the culture methodology, as gonococci are exceedingly sensi-
tive to external environmental factors.
In settings with more resources, NAATs have rapidly replaced
culture for detection of gonococci. NAATs have many advantages,
e.g., they detect nonviable gonococci; have a sensitivity superior to
those of all other diagnostic methods, particularly for pharyngeal
and rectal specimens; and are less demanding regarding specimen
collection (noninvasive, self-collected samples, such as urine
[males] and vaginal swabs [females], can effectively be used),
transportation, and storage. NAATs also are rapid, allow automa-
tion, and enable simultaneous detection of several pathogens.
However, NAATs also have disadvantages, e.g., they do not allow
AMR testing, the appropriate time for test of cure is still debated,
and the commercially available and in-house NAATs show differ-
ent sensitivities and particular specificities in their detection of N.
gonorrhoeae. Commensal Neisseria species, frequently present in
the pharynx and rectum but also, more rarely, in the urogenital
tract, have genetic homology with N. gonorrhoeae and might
cross-react in gonococcal NAATs, resulting in false-positive re-
ports. The suboptimal specificities of gonococcal NAATs result in
low positive predictive values (PPV), particularly in low-preva-
lence populations (17,20–24). In Europe and Australia, if the PPV
obtained using NAAT is 90% in the local setting, it is recom-
mended that a supplementary NAAT (with a different target se-
quence) be used for verification of all NAAT-screening-positive
samples (17,25). Importantly, in settings using only NAATs for
detection of gonococci, it is essential for the laboratories, epi-
demiologists, and clinicians to be involved in and aware of an
adequate local, national, and/or international gonococcal antimi-
crobial surveillance program (GASP). It is important to note that
immunofluorescence assays, enzyme immunoassays, and rapid
point-of-care tests for antigen or antibody detection with suffi-
cient specificity and, particularly, sensitivity for diagnosis are not
available for clinical diagnostic purposes.
Detection of Antimicrobial Resistance in N. gonorrhoeae
The quantitative agar dilution method determines the MICs (g/
ml) of antimicrobials and is the “gold standard” method. Never-
theless, particularly for testing small numbers of isolates, this
method is laborious and not ideal for routine AMR testing. Con-
sequently, the quantitative Etest method for MIC determination,
which is comparable to the agar dilution method, is frequently
utilized. Furthermore, a qualitative AMR determination can be
obtained with a disc diffusion assay. However, for adequate repro-
ducibility and interpretation to appropriately reflect the MIC val-
ues of given antimicrobials, these methods require considerable
quality assurance and appropriate quality controls. Disc diffusion
methods are recommended for use only when MIC determination
cannot be performed, due to limited resources or other reasons,
and any new, emerging, or rare AMR should be confirmed by MIC
determination (20).
For AMR testing, culture and phenotypic AMR testing remain
crucial. However, in many lower-resource settings, diagnosis of
gonorrhea depends upon syndromic management of patients, and
no specimens are taken. In many higher-resource settings, NAATs
have rapidly replaced culture for detection of N. gonorrhoeae. For
enhanced AMR surveillance, it is essential to strengthen the cul-
ture capacity globally. However, it is also imperative to develop
rapid genetic AMR testing (5,14,26–28). These methods should
ideally be used at point of care simultaneously with a rapid, sen-
sitive, and specific genetic test for gonococcal detection. These
methods could directly provide a diagnosis and guide individually
tailored treatments, ensuring rational antimicrobial use and af-
fecting the control of both gonorrhea and AMR. Mathematical
modeling can then explore the impact of NAAT-based AMR tests
on the spread of resistance and on clinical outcomes. Dynamic
transmission models can capture the net effects of competing fac-
tors, such as increased detection and treatment of gonorrhea, in-
creased reinfection risk, and reduced or delayed detection of AMR
upon gonorrhea transmission and of AMR-resistant strains (26).
Unfortunately, no commercially available gonococcal NAATs de-
tect any AMR determinants. However, in-house molecular assays
exist for detection of one or more genetic AMR resistance deter-
minants involved in plasmid-mediated penicillin resistance (29–
31), chromosomally mediated penicillin resistance (26,32–35),
Antimicrobial Resistance Expressed by N. gonorrhoeae
July 2014 Volume 27 Number 3 cmr.asm.org 589
plasmid- and chromosomally mediated tetracycline resistance
(36), resistance to macrolides (32,37–42), fluoroquinolone resis-
tance (43–49), ESC resistance (50–53), and multidrug resistance
(54–56). Unfortunately, for most AMR determinants, the sensi-
tivity and specificity of these molecular AMR assays for determi-
nation of AMR are often low. For example, for the currently rec-
ommended antimicrobials, the ESCs, the correlates between most
described resistance determinants, the ESC MICs of the gonococ-
cal strains, and the treatment outcome are highly suboptimal (5).
The ongoing evolution of ESC resistance, involving many differ-
ent mutations in several divergent genes, is a major challenge for
the development of a genetic AMR test for ESCs. Tests that need
continual updating with new target sequences are unlikely to be
profitable for companies manufacturing NAATs in the short term.
Some “strain-specific” molecular assays that detect mutations in-
volved in ESC resistance in the described XDR gonococcal strains
were also recently developed (57,58).
HISTORY OF TREATMENT REGIMENS AND RESISTANCE
EVOLUTION
The Preantimicrobial Era
During the preantimicrobial era, treatment of gonorrhea con-
sisted mainly of living a healthier lifestyle, with fresh air, appro-
priate food, and rest, abstaining from alcohol and sexual activity,
and receiving systemic treatment with different types of balsams,
urethral irrigations, chemical compounds, and hyperthermia
(59). During the second half of the 19th century, gonorrhea was
frequently treated with an Indonesian type of pepper (cubebs) and
with balsam extracted from a tree in South America (copaiba)
(60). To mask the taste and reduce toxicity, copaiba was fre-
quently mixed with licorice, magnesium hydroxide, or ammo-
nium carbonate or incorporated into gelatin capsules (61). An-
tiphlogistic regimens, avoiding irritation, maintaining the body
cool (using salts), and dilution of the urine could also be used until
inflammatory symptoms diminished, and then cubebs or copaiba
balsam was used three times a day (62). Soap and water enemas,
oral laudanum (opium tincture), and warm baths were used to
ease retention of urine, and if needed, catheterization was per-
formed. Acute urethritis could also be treated by anterior urethral
irrigation with dilutions of warm potassium permanganate for
several weeks (59,63).
In the late 1800s, the search for more specific antibacterial com-
pounds was initiated, and many metallic compounds, e.g., com-
pounds of arsenic, antimony, bismuth, gold, silver, and mercury,
were investigated. During World War I, soldiers were provided
prophylactic packages comprising condoms, calomel (mercuryl
chloride) ointment, and Argyrol/Protargol (silver compounds),
and postcoital treatment centers, including urethral irrigation fa-
cilities, were also used (59,63). Mercury compounds were later
frequently used; for instance, Mercurochrome-220 was used as a
urinary tract antiseptic (64). By mixing 1% mercurochrome in a
50% glucose solution, the injection became safer and more effica-
cious (65). Subsequently, in addition to intravenous mercuro-
chrome, a silver-protein complex or mercurochrome was instilled
into the urethra, or the seminal vesicles were irrigated with potas-
sium permanganate (66).
Diathermy or hyperthermia was also used early in several set-
tings. Initially, only inflamed joints in patients with gonococcal
arthritis were heated. However, when some arthritis cases re-
sponded only with the addition of genital hyperthermia, genitalia
also began to be treated (67,68). A fever cabinet was used, with
only the head outside, and temperatures above 41°C were main-
tained for 4 to 6 h. For cure, usually 5 or 6 treatments, provided
every third day, were required (69). Up to 80 to 90% of gonococcal
arthritis cases could be cured (70). Pretreatment with mercuro-
chrome in hypertonic glucose was later shown to increase the
efficacy of hyperthermia (71). Finally, fewer hyperthermia treat-
ments were usually required with the addition of pelvic heating,
i.e., insertion of heating elements for about 2 h into the rectum in
men and the vagina in women (sometimes also the rectum), re-
sulting in local temperatures of up to 44°C (72,73). Hyperthermia
was considered the best treatment for gonococcal arthritis and,
most commonly, also resolved any genital symptoms (73).
The Antimicrobial Era
The history of discovered and introduced antimicrobials and the
evolution of resistance, including genetic resistance determinants,
as well as changes in the recommended first-line antimicrobial(s),
are summarized in Fig. 1.
Sulfonamides. In 1935, Gerhard Domagk discovered sulfanil-
amide (59,63). The sulfonamides were the first antimicrobials
used for treatment of gonorrhea; sulfanilamide initially cured 80
to 90% of gonorrhoea cases (74–76). Sulfapyridine became avail-
able in 1940 to 1941, and a 1-week course of sulfapyridine could
cure many cases where sulfanilamide had failed (77). The subse-
quent drug sulfathiazol was as effective as sulfapyridine but was
more tolerable (76,78). Unfortunately, by 1944, many gonococcal
strains showed clinical resistance, and by the late 1940s, 90% of
gonococcal isolates were resistant to sulfonamides in vitro (75,
79). Sulfonamides (e.g., sulfamethoxazole) continued to be used,
particularly in combination with trimethoprim and in low-re-
source settings, for many decades (19,63,80,81).
Penicillin. In 1928, Alexander Fleming accidentally discovered
that a compound produced by a fungus could lyse staphylococci
and other bacteria causing many infectious diseases. He identified
that the fungus belonged to the Penicillium genus, and after ini-
tially being called “mold juice,” the compound was named peni-
cillin in early 1929. In 1930, Cecil Paine used a crude extract from
the penicillin-producing fungus Penicillium notatum to cure
gonococcal ophthalmia in an infant (82). However, it took until
1943 before this “wonder drug” was appropriately documented to
be therapeutically effective for gonococcal urethritis, and penicil-
lin subsequently marked a new era in the treatment of gonorrhea
as well as other infectious diseases (83,84). Penicillin quickly sup-
planted the sulfonamides as the first-line treatment of gonorrhea
(83,85). Penicillin cured more than 95% of cases, with total doses
as low as 45 mg being used (85). However, over time, the MICs of
penicillin against gonococcal strains increased due to an accumu-
lation of chromosomal resistance determinants, and the pre-
scribed doses were progressively increased to obtain appropriate
cure rates (8,63,86–89). Thus, by 1946, four gonorrhea cases
resistant to “high” doses of penicillin (0.6 to 1.6 million units)
were reported, and this resistance was also verified by in vitro
testing. A gradual increase in the proportion of gonococcal strains
with increasing resistance to penicillin was observed during the
two subsequent decades (90,91). Despite this developing situa-
tion, penicillin remained an effective antimicrobial for treatment
of gonorrhea for many decades. Nevertheless, after the “epidemic”
of gonorrhea in the United States and many other countries asso-
Unemo and Shafer
590 cmr.asm.org Clinical Microbiology Reviews
ciated with the “sexual revolution” of the 1960s, the level of pen-
icillin required to treat uncomplicated gonorrhea had substan-
tially increased, and treatment failures were reported (8,89,92).
The emergence in 1976 of two types of -lactamase-encoding
plasmids which caused high-level resistance to penicillin, origi-
nating in Southeast Asia and sub-Saharan West Africa, in certain
gonococcal strains from the United States and the United King-
dom (93–95) reinforced the fear that the decades-long use of pen-
icillin might soon end. The rapid international spread of these
strains was of great concern. However, when penicillin was aban-
doned as a first-line antimicrobial in the United States and several
other countries about a decade later, the primary reason was the
emergence of chromosomally mediated clinical resistance to pen-
icillin. An outbreak of chromosomally mediated penicillin-resis-
tant gonorrhea in Durham, NC (96,97), was the first major blow
to the continued use of penicillin. Currently, gonococcal strains
with plasmid- and/or chromosomally mediated resistance to pen-
icillin are common globally (5,8,19,81,98–106).
Tetracycline. The first tetracycline, chlortetracycline (aureo-
mycin), was discovered in an allotment soil bacterium in 1945 by
Benjamin Minge Duggar. Tetracyclines were used early to treat
gonorrhea, especially in patients with penicillin allergy. However,
the MICs of tetracycline against gonococcal strains increased over
time, due to chromosomal resistance determinants (88). The
emergence of the tetM determinant (causing high-level tetracy-
cline resistance) on the conjugative plasmid in the mid-1980s
(107) resulted in the exclusion of tetracycline from treatment
guidelines in the United States and many countries worldwide.
These gonococcal strains with plasmid-mediated high-level resis-
tance to tetracyclines were first reported in 1986 in the United
States and soon thereafter in the Netherlands (108) and are now
widespread internationally (8,19,81,98–104).
Spectinomycin. In the early 1960s, spectinomycin was synthe-
sized and commercialized as a specific gonorrhea treatment. Spec-
tinomycin is an aminocyclitol that is closely related to the amin-
oglycosides produced by Streptomyces spectabilis. Spectinomycin
is produced in nature by many organisms, including cyanobacte-
ria. After the emergence of plasmid-mediated high-level resistance
to penicillin, spectinomycin was frequently used for treatment of
these cases (109,110). Nevertheless, in 1967, spectinomycin resis-
tance was reported for a penicillin-susceptible gonococcal strain
in the Netherlands (111), and in 1981, a spectinomycin-resistant
gonococcal isolate with plasmid-mediated high-level resistance to
penicillin was reported in the Philippines (112). In 1981 in South
Korea, spectinomycin was introduced as a first-line gonorrhea
treatment in U.S. military personnel. However, after only 4 years,
a clinical failure rate of 8.2% was described (113). Furthermore, in
1983, many spectinomycin-resistant gonococcal isolates were re-
ported from London, United Kingdom (114). Subsequently, spec-
tinomycin was abandoned as a first-line empirical monotherapy
for gonorrhea internationally. Currently, spectinomycin resis-
tance, particularly high-level resistance, is exceedingly rare in
gonococcal strains worldwide. However, spectinomycin is cur-
rently not available and used in many countries, and it is feared
that resistance will be selected rapidly if spectinomycin is intro-
duced for first-line treatment. Furthermore, spectinomycin is
suboptimal for treatment of pharyngeal gonorrhea, i.e., its efficacy
rate is around 80% (115–117).
FIG 1 Historyof discovered and recommended antimicrobials and evolution of resistance in Neisseria gonorrhoeae, including the emergence of genetic resistance
determinants, internationally. During the preantimicrobial era (before the 1930s), treatment consisted of, e.g., a healthier lifestyle, copaiba, cubebs, urethral
irrigations, potassium permanganate, silver compounds, mercury compounds, and hyperthermia. SUL, sulfonamides; PEN, penicillin; SPT, spectinomycin;
TET, tetracycline; CIP, ciprofloxacin; OFX, ofloxacin; CFM, cefixime; CRO, ceftriaxone; AZM, azithromycin; DOX, doxycycline.
Antimicrobial Resistance Expressed by N. gonorrhoeae
July 2014 Volume 27 Number 3 cmr.asm.org 591
Quinolones. Synthetic quinolone antimicrobials were discov-
ered by George Lesher and colleagues as a by-product of the man-
ufacture of chloroquine in the 1960s, and the quinolone nalidixic
acid was introduced for treatment of urinary tract infections in
humans. Nalidixic acid is the predecessor of all quinolones, and
subsequent, broader-spectrum quinolones are known as fluoro-
quinolones. The fluoroquinolones ciprofloxacin and ofloxacin
were previously recommended for gonorrhea treatment, and cip-
rofloxacin in particular was widely used to treat gonorrhea from
the mid- to late 1980s onwards. Initially, low doses, e.g., 250 mg, of
ciprofloxacin were used, but clinical failures were already reported
by 1990 (118). The recommended ciprofloxacin dose was raised to
500 mg, but resistance developed and spread quickly, initially in
the Asian Western Pacific Region (119,120). In some Asian West-
ern Pacific countries, fluoroquinolones were abandoned as first-
line empirical treatments of gonorrhea by the mid- to late 1990s
(8). Ciprofloxacin-resistant gonococcal strains were subsequently
rapidly exported internationally or emerged independently (121–
123). In regard to the United States, in 2000, fluoroquinolone-
resistant strains initially imported from Asia were prevalent in
Hawaii (124), and subsequently, these strains spread first to the
West Coast and then to the rest of the United States, predomi-
nantly among MSM (125). In 2007, the fluoroquinolones were
abandoned from the CDC-recommended treatment regimens for
gonorrhea, with no exceptions (126). Due to high levels of fluo-
roquinolone resistance, many Asian and European countries re-
moved ciprofloxacin as a first-line treatment in the early to mid-
2000s (8). Currently, the prevalences of fluoroquinolone-resistant
gonococcal strains are high worldwide (5,8,19,63,81,98–106).
Macrolides. In 1952, the macrolides were discovered when
erythromycin was isolated from the soil microorganism Strepto-
myces erythraeus, currently known as Saccharopolyspora erythraea.
In 1980, azithromycin, a synthetic derivative of erythromycin, was
developed. Clinical and in vitro AMR data showed early that eryth-
romycin is not sufficiently effective for the treatment of gonorrhea
(63,127). Compared to erythromycin, azithromycin has a sub-
stantially higher activity against N. gonorrhoeae. However, by the
mid- to late 1990s, decreased susceptibility and resistance to azi-
thromycin were reported from Latin America, where azithromy-
cin was frequently used early on for treatment of bacterial STIs,
including gonorrhea (99,128,129). Subsequently, azithromycin
resistance emerged in many countries, particularly where there
was a high level of azithromycin usage for treatment of gonorrhea
and also, e.g., for Chlamydia trachomatis infections (99,102,130,
131). Worryingly, gonococcal isolates with high-level resistance to
azithromycin (MICs of 256 g/ml) were identified in Scotland
(132), England (37), Argentina (38), Italy (133), the United States
(39), and Sweden (42). Despite being used in several countries,
azithromycin is not recommended for empirical monotherapy of
gonorrhea. This is due particularly to the concerns of a rapid se-
lection of resistance but also to the possible adverse events from
taking the 2-g azithromycin oral dose (37,134,135). Nevertheless,
azithromycin is one of the two antimicrobials in all the introduced
dual-antimicrobial therapeutic regimens for gonorrhea (15–17).
Cephalosporins. The first cephalosporin compounds were iso-
lated from cultures of the fungus Cephalosporium acremonium,
first discovered by Giuseppe Brotzu in 1948. Chemical modifica-
tions of these and similar compounds resulted in the first useful
antimicrobial agent, i.e., cefalotin, launched in 1964. The cepha-
losporins most commonly recommended internationally for
treatment of gonorrhea following the demise of fluoroquinolones
are the third-generation ESCs ceftriaxone (injectable) and ce-
fixime (oral). No other injectable or oral ESCs have any evident
advantages over ceftriaxone and cefixime (17,63,136). Neverthe-
less, other oral cephalosporins have been used when cefixime has
not been available, e.g., cefditoren and celdinir in Japan, cefu-
roxime in several European countries, cefpodoxime in the United
States, and ceftibuten in Hong Kong (63,136–138). During the
last 2 decades, gonococcal strains exhibiting resistance to ESCs
seem to have initially emerged in Japan and then spread world-
wide. In Japan, ceftriaxone was not endorsed for gonorrhea treat-
ment from the 1990s to the early 2000s. Consequently, many oral
cephalosporins and dose regimens, including some with subopti-
mal efficacies, were prescribed for monotherapy, but if resistance
was identified, cefodizime or spectinomycin was administered
(139,140). Multiple low-dose regimens of oral cephalosporins
were frequently used, which could have resulted in subinhibitory
cephalosporin concentrations and, accordingly, may have selected
for cephalosporin resistance (5,140–143). Furthermore, when
single-dose cefixime (the most potent oral ESC) therapy was ap-
plied in Japan, it commonly included only 300 mg of cefixime, in
contrast to the 400-mg dose used internationally (5,144). Thus,
between 1995 and 2000, in Fukuoka, Japan, the MIC peaks for
cefixime and ceftriaxone against gonococcal isolates reached 0.25
g/ml and 0.064 g/ml, respectively (140). Furthermore, between
1999 and 2002, in six hospitals in central Japan, the proportions of
gonococcal isolates with in vitro resistance to cefixime (MICs of
0.5 g/ml) and ceftriaxone (MICs of 0.5 g/ml) reached
30.2% and 0.9%, respectively (143). This also translated into treat-
ment failures with cefixime. Accordingly, from 1999 to 2001, eight
treatment failures with cefixime (200 mg orally twice, 6 h apart)
were reported (142), and in 2002 to 2003, four treatment failures
with an extended cefixime regimen (200 mg orally twice a day for
3 days) were documented (145). In 2006, all oral ESCs were ex-
cluded from the treatment guidelines in Japan, and since then,
ceftriaxone (1 g intravenously), which is mostly used, cefodizime
(1 g intravenously), and spectinomycin (2 g intramuscularly) have
been recommended as first-line empirical treatments for uncom-
plicated anogenital and pharyngeal gonorrhoea (146). During the
last decade, strains with decreased susceptibility or resistance to
ESCs spread internationally, and their presence has been docu-
mented basically globally (5,8,98–100,102,104–106,131,147–
151). Worryingly, gonococcal AMR surveillance remains highly
limited in many regions worldwide (104,106,152), and accord-
ingly, the global burden of decreased susceptibility and resistance
to ESCs is largely unknown. Currently, cefixime treatment failures
have been verified in Japan, several European countries, Canada,
and South America (4,142,145,153–157), and a few ceftriaxone
treatment failures for pharyngeal gonorrhea have been identified
in Japan, some European countries, and Australia (3,158–161).
It is of great concern that the first gonococcal XDR strains,
exhibiting high-level clinical resistance to all ESCs combined with
resistance to nearly all other available therapeutic antimicrobials,
were recently identified in Kyoto, Japan (3), Quimper, France (4),
and Catalonia, Spain (2). All these XDR strains were also identi-
fied in high-risk, frequently transmitting populations, i.e., com-
mercial sex workers (CSWs) or MSM. Since ceftriaxone is the last
option for first-line empirical monotherapy of gonorrhea, the
emergence of XDR gonococci might initiate an era of gonorrhea
that is untreatable using antimicrobial monotherapy. Fortunately
Unemo and Shafer
592 cmr.asm.org Clinical Microbiology Reviews
for now, however, based on the intensified surveillance under-
taken in Kyoto and Osaka (2010 to 2012) after identification of the
first XDR strain (H041), this strain has not spread further within
the local community (162), which might indicate a lowered bio-
logical fitness.
MECHANISMS OF ANTIMICROBIAL RESISTANCE IN N.
GONORRHOEAE
Resistance Emergence and Spread
N. gonorrhoeae has an extraordinary capacity to alter its genetic
material, given that it is naturally competent for transformation
(transfer of partial or whole genes) during its entire life cycle and
because it can effectively change its genome through all types of
mutations. N. gonorrhoeae uses these mechanisms to rapidly adapt
to and survive in the often hostile environments at different sites
in the human host, and accordingly, the bacterium is a great ex-
ample of “survival of the fittest.” The gonococcus has in this way
evolved and acquired or developed nearly all known physiological
mechanisms of AMR to all antimicrobials recommended and/or
used for treatment, e.g., (i) antimicrobial destruction or modifi-
cation by enzymatic means, (ii) target modification or protection
that reduces affinity for the antimicrobials, (iii) decreased influx
of antimicrobials, and (iv) increased efflux of antimicrobials.
Most genetic AMR determinants in N. gonorrhoeae are situated
chromosomally, and only the bla
TEM
gene (93,95) and the tetM
gene (107), which result in high-level resistance to penicillin and
tetracycline, respectively, are known to be plasmid borne in gono-
cocci. Certain AMR determinants alone can result in high-level
resistance in vitro and in vivo, i.e., treatment failure, for the anti-
microbial in question. However, in other instances, acquisition of
a single AMR determinant confers only an incremental increase in
AMR that is of less clinical significance; nevertheless, the cumula-
tive effects of several AMR determinants and their complex inter-
actions and interplay can ultimately result in clinical levels of
AMR. For example, this is the scenario resulting in the demise of
penicillin as an effective treatment for gonorrhea, due to several
chromosomally mediated resistance determinants. The described
determinants and mechanisms of AMR in N. gonorrhoeae are
summarized in Table 1.
Gonococci develop AMR through gene transfer (transforma-
tion and subsequent recombination into the genome) or by spe-
cific mutations. Exposure of gonococci or other Neisseria spp. to
antimicrobials given for treatment of gonorrhea or other infec-
tions can select for resistant strains. The commensal Neisseria spp.
frequently inhabit human anatomical sites, particularly the phar-
ynx, and are often exposed to antimicrobials. Accordingly, resis-
tance can initially emerge in commensal Neisseria spp. that act as a
reservoir of AMR genes, which can readily be transferred to gono-
cocci through transformation. Likely, the mostly asymptomatic
pharyngeal gonorrhea, where gonococci and commensal Neisseria
spp. can coexist for extended periods, provides the means for this
gene transfer (5,8,19,163–166). Horizontal gene transfer most
probably played a pivotal role in the spread of mosaic penA alleles
(see “Cephalosporin Resistance”), resulting in decreased suscep-
tibility or resistance to ESCs (3,4,166). After their emergence,
AMR gonococcal strains can spread quickly, first within a geo-
graphical region and then later establishing an international pres-
ence. Furthermore, AMR genes can be spread between gonococcal
strains by transformation (of chromosomal or plasmid DNA) or
conjugal transfer of plasmid AMR genes. Gonococci use a se-
quence-specific DNA uptake system to recognize DNA from
themselves or closely related species (167,168). Chromosomal
DNA transformation frequencies in gonococci can be quite high
(10
2
/g DNA/10
8
CFU). However, plasmid transformation fre-
quencies are substantially lower (10
6
), and deletions occur fre-
quently (169). Thus, even though the rate of spontaneous mis-
sense mutations that result in AMR can be low, horizontal transfer
of these alleles by transformation is very efficient in disseminating
AMR within the community.
Antimicrobials most frequently initiate their activity through
binding to a specific target that is critical for viability of a bacte-
rium. By this binding, the antimicrobials block the bacterium’s
function and the microbe succumbs. However, bacteria can de-
velop low- to high-level resistance through mutations that reduce
or abrogate antimicrobial binding to the specific target. Because
these targets are critical for cell viability, the changes that occur
must remodel the active site of the target to lower its affinity for the
antimicrobial without greatly affecting the normal function of the
enzyme and having a detrimental impact on bacterial physiology
and fitness. In N. gonorrhoeae, most of the acquired or developed
AMR mechanisms do not appear to cause significantly lower bio-
logical fitness (possibly mainly due to compensatory mutations),
which results in the persistence of AMR and MDR/XDR strains
even in the absence of obvious antimicrobial selection. In fact,
some AMR determinants can enhance the biological fitness of
specific gonococcal strains (170–172). Consequently, the prospect
of being able to use previously withdrawn antimicrobials for gon-
orrhea treatment appears bleak (5,8).
Sulfonamide Resistance
Sulfonamides target the bacterial dihydropteroate synthase
(DHPS) enzymes, thereby inhibiting the synthesis of folic acid in
the bacterium. Sulfonamide resistance can be mediated by over-
synthesis of p-aminobenzoic acid, which dilutes the antimicrobial
agent, or by alterations in the folP gene (point mutations or the
presence of a mosaic gene containing DNA sequences from com-
mensal Neisseria spp.), encoding the drug target DHPS. The alter-
ations of DHPS result in a significantly lowered affinity for the
sulfonamide agents and a bacteriostatic activity (173–175).
Penicillin Resistance
Plasmid-mediated penicillin resistance. -Lactam antimicrobi-
als, such as penicillins and cephalosporins, inhibit the formation of
peptidoglycan cross-links in the bacterial cell wall through binding of
the -lactam ring to transpeptidase enzymes (penicillin-binding pro-
teins [PBPs]), which results in bactericidal activity.
Gonococcal strains with plasmid-mediated high-level resis-
tance to penicillin traditionally contain plasmids with a bla
TEM-1
gene, encoding a TEM-1-type -lactamase. This enzyme hydro-
lyzes the cyclic amide bond of -lactamase-susceptible penicillins,
opening the -lactam ring and rendering the penicillin inactive.
The gonococcal -lactamase plasmids were likely acquired by
conjugal transfer from Haemophilus parainfluenzae (176,177),
which can carry a closely related R plasmid, RSF0885. After the
first descriptions of gonococcal strains with -lactamase-produc-
ing plasmids in 1976 (93,95), these strains and the plasmids them-
selves (between gonococcal strains) spread rapidly internation-
ally. Currently, gonococcal strains possessing the Asian (7,426 bp)
and African (5,599 bp) plasmids (named after their epidemiolog-
Antimicrobial Resistance Expressed by N. gonorrhoeae
July 2014 Volume 27 Number 3 cmr.asm.org 593
ical origins) are globally widespread (19,81). However, other
types of -lactamase-producing plasmids have been described for
gonococci, some of which are also prevalent, including the To-
ronto (5,153 bp), Rio (5,153 bp; possibly identical to Toronto),
Nîmes, New Zealand, and Johannesburg plasmids. The Asian
plasmid appears to be the ancestral plasmid from which the other
plasmids evolved, through deletions and/or insertions. Accord-
ingly, these -lactamase-producing plasmids may be character-
TABLE 1 Resistance determinants and mechanisms in Neisseria gonorrhoeae for antimicrobials previously or currently recommended for treatment
of gonorrhea
Antimicrobial class Resistance determinants/mechanisms
Sulfonamides Oversynthesis of p-aminobenzoic acid, which dilutes the sulfonamide.
Mutations in folP (encoding the sulfonamide target DHPS) reduce target affinity. The folP mutations comprise
SNPs or a mosaic folP gene containing sequences from commensal Neisseria spp.
Penicillins (e.g., penicillin G and
ampicillin)
Mutations in penA (encoding the main lethal target PBP2). Traditionally, the mutations were the single amino
acid insertion D345 in PBP2 and 4 to 8 concomitant mutations in the PBP2 carboxyl-terminal region,
decreasing the PBP2 acylation rate and reducing susceptibility 6- to 8-fold. In the last decade, many mosaic
penA alleles with up to 70 amino acid alterations, also reducing PBP2 acylation, were described.
Mutations in mtrR, in the promoter (mainly a single nucleotide [A] deletion in the 13-bp inverted repeat
sequence) or coding sequence (commonly a G45D substitution), result in overexpression of and increased
efflux from the MtrCDE efflux pump. See the text for rarer mutations resulting in increased MtrCDE efflux.
porB1b SNPs, e.g., encoding G120K and G120D/A121D mutations in loop 3 of PorB1b, reduce influx (penB
resistance determinants). Interestingly, the penB phenotype is apparent only in strains with the mtrR
resistance determinant.
A SNP in pilQ (encoding the pore-forming secretin PilQ of the type IV pili), i.e., E666K, reduces influx. Note
that this SNP has been found only in the laboratory and is unlikely to be present in clinical isolates, because it
disrupts type IV pilus formation, which is essential for pathogenesis.
A SNP in ponA (encoding the second penicillin target, PBP1), i.e., “ponA1 determinant” (L421P), reduces
penicillin acylation of PBP1 2- to 4-fold.
“Factor X,” an unknown, nontransformable determinant, increases penicillin MICs 3- to 6-fold.
Penicillinase (TEM-1 or TEM-135)-encoding plasmids, i.e., Asian, African, Toronto, Rio, Nîmes, New Zealand,
and Johannesburg plasmids, hydrolyze the cyclic amide bond of the -lactam ring and render the penicillin
inactive.
Tetracyclines (e.g., tetracycline
and doxycycline)
A SNP in rpsJ (encoding ribosomal protein S10), i.e., V57M, reduces the affinity of tetracycline for the 30S
ribosomal target.
mtrR mutations (see above).
penB mutations (see above).
A SNP in pilQ (see above).
TetM-encoding plasmids, i.e., American and Dutch plasmids. Evolved derivatives have been described in
Uruguay and South Africa. TetM, resembling elongation factor G, binds to the 30S ribosomal subunit and
blocks tetracycline target binding.
Spectinomycin A 16S rRNA SNP, i.e., C1192U, in the spectinomycin-binding region of helix 34, reduces the affinity of the drug
for the ribosomal target.
Mutations in rpsE (encoding the 30S ribosomal protein S5), i.e., the T24P mutation and deletions of V25 and
K26E, disrupt the binding of spectinomycin to the ribosomal target.
Quinolones (e.g., ciprofloxacin
and ofloxacin)
gyrA SNPs, e.g., S91F, D95N, and D95G, in the QRDR, reduce quinolone binding to DNA gyrase.
parC SNPs, e.g., D86N, S88P, and E91K, in the QRDR, reduce quinolone binding to topoisomerase IV.
Many additional mutations in the QRDR of gyrA and parC have been described. An overexpressed NorM efflux
pump also slightly enhances quinolone MICs.
Macrolides (e.g., erythromycin
and azithromycin)
23S rRNA SNPs, i.e., C2611T and A2059G (in 1 to 4 alleles), result in a 23S rRNA target (peptidyltransferase
loop of domain V) with a reduced affinity for the 50S ribosomal macrolide target.
mtrR mutations (see above).
erm genes (ermB,ermC, and ermF), encoding rRNA methylases that methylate nucleotides in the 23S rRNA
target, block the binding of macrolides.
MacAB efflux pump; its overexpression increases the MICs of macrolides.
mef-encoded efflux pump exports macrolides out of the bacterial cell and increases the MICs of macrolides.
Cephalosporins (e.g., ceftibuten,
cefpodoxime, cefixime,
cefotaxime, and ceftriaxone)
Mosaic penA alleles encoding mosaic PBP2s with a decreased PBP2 acylation rate. These proteins have up to 70
amino acid alterations and are derived from horizontal transfer of partial penA genes from mainly commensal
Neisseria spp. Mutations in mosaic PBP2s verified to contribute to resistance are A311V, I312M, V316T,
V316P, T483S, A501P, A501V, N512Y, and G545S. The resistance mutations need other epistatic mutations
in the mosaic penA allele.
penA SNPs, i.e., A501V and A501T, in nonmosaic alleles can also enhance cephalosporin MICs. Some additional
SNPs (G542S, P551S, and P551L) were statistically associated with enhanced cephalosporin MICs, but their
effects remain to be proven with, e.g., site-directed penA mutants in isogenic backgrounds.
mtrR mutations (see above).
penB mutations (see above).
“Factor X,” an unknown, nontransformable determinant (see above).
Unemo and Shafer
594 cmr.asm.org Clinical Microbiology Reviews
ized as either deletion derivatives of the Asian plasmid (Africa,
Toronto, Rio, and Johannesburg plasmids) or insertion deriva-
tives of either the Asian (New Zealand plasmid) or African (Nîmes
plasmid) plasmid (30,178–182). All these plasmids likely contain
aTnA(Tn2) transposable element carrying the bla
TEM-1
gene that
encodes TEM-1 -lactamase. No extended-spectrum -lactamase
(ESBL) has yet been acquired or developed in gonococci. Never-
theless, in many currently circulating strains, the bla
TEM-135
gene,
which differs by one single nucleotide polymorphism (SNP) from
bla
TEM-1
, has been found, and only one additional SNP could re-
sult in an ESBL capable of hydrolyzing and degrading ESCs (183–
185).
Chromosomally mediated penicillin resistance. Chromo-
somally mediated penicillin resistance in gonococci is due to mu-
tations that modify the target proteins (PBPs), in complex inter-
actions and in an interplay with resistance determinants that
increase the efflux and decrease the influx of penicillin (see be-
low).
The target molecules for -lactam antimicrobials, i.e., trans-
peptidases (PBPs), contain three conserved motifs in their active
sites: the SxxK, SxN, and KTG motifs. In penicillin-resistant gono-
cocci, traditionally there have been 5 to 9 mutations in the penA
gene (encoding PBP2, the main lethal target for -lactam antimi-
crobials), and together, these decrease the acylation rates of PBP2
and, accordingly, decrease the susceptibility to penicillin 6- to
8-fold (186–188). These penA mutations were acquired by gono-
cocci through transformation of penA sequences from commensal
Neisseria spp. that possess a PBP2 with a reduced rate of acylation
by penicillin (189–192). The most common PBP2 mutation in
penicillin-resistant gonococcal strains has traditionally been in-
sertion of an aspartate (named Asp345a), and the remaining mu-
tations lie in the carboxyl-terminal region of PBP2 (193). The
structures of wild-type PBP2 and PBP2 containing four C-termi-
nal mutations found in the penicillin-resistant strain FA6140 were
recently published (186). Asp345a is located on a -hairpin loop
(2a to 2d) close to the active site, and the C-terminal mutations
are also relatively close to the active site (186,194). Although the
C-terminal mutations significantly affect rates of acylation by
penicillin, the crystal structure of PBP2 is not altered (186), which
is consistent with the necessity for the mutated PBP2 enzyme to
retain activity with its natural substrate. In the absence of a crystal
structure of PBP2 containing the Asp345a insertion, the impact of
this mutation is not totally evident, but most likely it is more
significant than the C-terminal substitutions. Notably, only an
aspartate insertion confers resistance (195), and consistent with
this, only an aspartate insertion is observed in clinical gonococcal
strains (190). This might suggest that only an insertion of aspar-
tate, not closely related amino acids, such as glutamate or aspara-
gine, can discriminate against -lactam antimicrobials without
abolishing the PBP2 transpeptidase activity essential for viability
(196). During the latest decade, many mosaic penA genes have also
been described. These mosaic genes contain up to 60 to 70 amino
acid changes compared to a wild-type penA gene and can result in
resistance to both penicillins and ESCs (2–5). For details regarding
these genes, see “Cephalosporin Resistance.”
Although PBP2 alteration is the primary mechanism for chro-
mosomally mediated penicillin resistance in gonococci, strains
exhibiting high-level penicillin resistance also harbor a single mis-
sense mutation in the ponA gene (termed the ponA1 allele) that
encodes PBP1, which has an approximately 16-fold lower penicil-
lin acylation rate than that of wild-type PBP2 (187,196). The
ponA1 allele encodes a Leu421Pro alteration in PBP1, which re-
duces the rate of penicillin acylation of PBP1 3- to 4-fold (187).
The structural consequences of this mutation are unknown be-
cause of the lack of any crystal structure for PBP1. Interstingly, in
a penicillin-resistant strain, reversion of ponA1 to wild-type ponA
decreases the penicillin MIC 2- to 4-fold, but introduction of
ponA1 into a strain with penA,mtrR, and penB resistance determi-
nants (see below) does not affect the penicillin MIC (187). This
may indicate epistasis or the presence of some unknown resistance
determinant, such as “factor X” (see below).
Furthermore, penicillin MICs can be increased further by spe-
cific mutations resulting in increased efflux by overexpression of
the MtrCDE efflux pump system, which exports the penicillin out
of the cell (mtrR resistance determinant) (3–5,197–199), and by
mutations resulting in a decreased influx (intake) of penicillin,
through a decreased permeability of the outer membrane channel
porin PorB1b (penB resistance determinants) (3–5,199–201). In
laboratory isolates, specific mutations in pilQ (encoding loss-of-
function alterations, e.g., E666K, in the pore-forming secretin
PilQ of type IV pili) are also found in strains with high-level resis-
tance to penicillin that contain alterations of penA,mtrR, and penB
(5,187,199,202). However, these pilQ mutations are unlikely to
be found in clinical isolates because they disrupt proper formation
of the type IV pili, which are essential for pathogenesis of gono-
cocci (203). Details regarding increased efflux and decreased in-
flux of antimicrobials are described below. Finally, there remains
at least one unknown, nontransformable penicillin resistance de-
terminant, “factor X,” which can increase the MICs of penicillin
approximately 3- to 6-fold (3–5,204).
Tetracycline Resistance
Plasmid-mediated tetracycline resistance. Tetracyclines inhibit
the binding of aminoacyl-tRNA to the mRNA-ribosome complex,
mainly by binding to the 30S ribosomal subunit, and accordingly
inhibit the protein synthesis that results in a bacteriostatic effect.
High-level plasmid-mediated resistance to tetracycline (MICs
of 16 g/ml) in gonococci is due to the tetM gene, initially de-
scribed for the Streptococcus genus (107). TetM confers high-level
resistance to tetracycline by binding to the ribosomes and causing
the release of the tetracycline molecule, thereby permitting pro-
tein synthesis to proceed. TetM achieves this by its resemblance to
elongation factor G (EF-G), involved in protein synthesis, and
TetM also has ribosome-dependent GTPase activity (205–207).
tetM initially established itself in gonococci by integrating into the
24.5-MDa conjugative plasmid to produce a 25.2-MDa (40.6 kb)
plasmid (208,209). Once established, it was stably maintained and
could be transferred to other gonococci by conjugation. The first
N. gonorrhoeae conjugative plasmid was identified in 1974 (210),
and this plasmid can also transfer -lactamase-producing plas-
mids between different gonococcal strains, and to Neisseria men-
ingitidis (211–213), Haemophilus influenzae, and Escherichia coli
(214). The tetM-possessing conjugative plasmid was first de-
scribed in 1985 in the United States and was designated the Amer-
ican tetM plasmid (107). In 1991, the highly homologous (215)
Dutch tetM plasmid was described (216). Subsequently, evolved
derivatives of these plasmids have been identified, e.g., the Uru-
guay (217) and South Africa (218) plasmids.
Chromosomally mediated tetracycline resistance. Chromo-
somally mediated tetracycline resistance in gonococci is due to
Antimicrobial Resistance Expressed by N. gonorrhoeae
July 2014 Volume 27 Number 3 cmr.asm.org 595
mutations that modify the ribosomal protein (target) structure, in
an interplay with resistance determinants that increase the efflux
and decrease the influx of tetracycline.
It was early shown that tetracycline-resistant gonococcal
strains had the so-called tet-2 mutation in addition to mtrR and
penB mutations (188). tet-2 was subsequently shown to be a mu-
tated rpsJ allele, which encoded an altered form of ribosomal pro-
tein S10 (219). The mutated rpsJ allele contained a SNP that al-
tered Val57 to Met57 in S10, and Leu57 and Gln57 substitutions
conferred identical levels of resistance. Val57 in S10 is located at
the vertex of a small loop near the aminoacyl-tRNA region that
forms the tetracycline-binding site, and it has been suggested that
replacement of the native Val57 with large uncharged amino acids
alter the rRNA structure, thereby reducing the affinity of tetracy-
cline for the ribosome (219).
In addition to these target modifications, as for penicillin (see
above), an increased efflux and decreased influx of tetracycline,
due to the mtrR resistance determinant (197–199) and penB resis-
tance determinants (199–201), respectively, result in an enhanced
resistance to tetracycline.
Spectinomycin Resistance
Spectinomycin binds to the 30S ribosomal subunit of the bacte-
rium and inhibits protein translation, resulting in a bacteriostatic
effect. Specifically, spectinomycin interacts with 16S rRNA and,
during polypeptide elongation, blocks the EF-G-catalyzed trans-
location of the peptidyl-tRNA from the A site to the P site. This
16S rRNA interaction is close to the base-paired nucleotides
G1064 –C1192 (E. coli numbering) in helix 34 (194,220).
For gonococci, high-level spectinomycin resistance (MICs of
1,024 g/ml) was early shown to be caused by a C1192U SNP in
the spectinomycin-binding region of helix 34 in 16S rRNA, in-
cluding the cross-linked positions 1063 to 1066 and 1190 to 1193
(221,222). Recently, a deletion of Val25 and a K26E alteration in
the 30S ribosomal protein S5, encoded by the rpsE gene, were also
verified to result in high-level spectinomycin resistance in gono-
cocci (223). A T24P mutation in S5 resulted in low-level spectino-
mycin resistance (MIC of 128 g/ml) (223,224). The N terminus
of S5, specifically amino acids 21 to 35, forms a loop that can bind
to helix 34 of 16S rRNA and is also involved in spectinomycin
binding to the ribosome (225). The alterations in ribosomal pro-
tein S5 likely disrupt its binding to 16S rRNA, which results in
spectinomycin resistance.
Quinolone Resistance
Bacterial DNA gyrase and topoisomerase IV are highly conserved
type II topoisomerases that are essential for DNA metabolism.
They act by breaking and rejoining double-stranded DNA in a
reaction that is coupled with ATP hydrolysis. Quinolones act by
inhibition of DNA gyrase and topoisomerase IV, resulting in bac-
tericidal activity.
Bacteria develop quinolone resistance through mutations that
alter quinolone recognition of these target enzymes. DNA gyrase
consists of a heterotetramer of two GyrA subunits and two GyrB
subunits; in gonococci, initial mutations in the primary target
gene, gyrA, are associated with resistance. The gyrA mutations
reduce quinolone binding affinity, rendering the enzyme (and the
bacteria) resistant to its inhibitory effect. Topoisomerase IV is a
tetramer of two ParC and two ParE subunits, encoded by the parC
and parE genes, respectively. The enzymatic activity of topoisom-
erase IV can also be inhibited by quinolones, although higher con-
centrations than those needed to inhibit DNA gyrase in vitro are
required. Specific SNPs in gyrA alone provide low- to intermedi-
ate-level resistance, but high-level resistance requires one or sev-
eral specific concomitant mutations in parC. These mutations can
easily be selected by exposure to subinhibitory ciprofloxacin con-
centrations and also transferred to other gonococci by transfor-
mation (226). Thus, a missense mutation at codon 91 in gyrA
(S91F), which is located within the so-called quinolone resistance-
determining region (QRDR) in E. coli GyrA, was shown to confer
a 100-fold increase in resistance to ciprofloxacin. Subsequent mu-
tation at codon 95 (D95N) further increased ciprofloxacin resis-
tance, by 2-fold. Higher levels of quinolone resistance required
mutations in parC in addition to those in gyrA. These parC muta-
tions mapped to codons 88 (S88P) and 91 (E91K) (226). Subse-
quently, additional GyrA/ParC amino acid alteration patterns
were identified in ciprofloxacin-resistant strains isolated interna-
tionally (227–229). Mutations in gyrB and parE do not appear to
have any significant impact on ciprofloxacin resistance (227,228,
230).
Macrolide Resistance
Macrolides inhibit protein synthesis by binding to the 50S ribo-
somal subunit, preventing translocation of the peptidyl-tRNA,
blocking the peptide exit channel in 50S subunits by interacting
with 23S rRNA, and causing ribosomes to release incomplete
polypeptides (231). This results in a bacteriostatic effect.
Bacterial resistance to macrolides may result from modification
of the ribosomal target by either rRNA methylase-associated
modification of 23S rRNA or specific mutations in 23S rRNA
and/or from an overexpressed efflux pump system. rRNA meth-
ylases can cause macrolide resistance through blocking of macro-
lide binding to 23S rRNA by methylating an adenosine residue at
position 2058 (E. coli numbering system), which is located in pep-
tidyl transferase domain V. Genes encoding rRNA methylase (re-
ferred to as macrolide-lincosamide-streptogramin B resistance
genes, or erm genes) can be carried by conjugative transposons,
and certain erm genes were identified in gonococcal strains in the
early 1990s (41). In gonococci, erm genes can confer high-level
resistance to erythromycin (MICs of 4 to 16 g/ml) and decreased
susceptibility or low-level resistance to azithromycin (MICs of 1 to
4g/ml) in the absence of other resistance determinants, such as
mtrR mutations or a mef-encoded efflux pump (see below) (41,
232). Clinical gonococcal isolates from the United States and Uru-
guay were reported by Roberts et al. (41) to contain the rRNA
methylase gene ermF or ermB/ermF.ermF was part of a complete
conjugative element, and its nucleotide sequence was 97% identi-
cal over 374 bp to ermF of Bacteroides fragilis.ermF could also be
transferred conjugally to other gonococci, meningococci, and En-
terococcus faecalis. Nevertheless, during recent years, erm genes
have been very rare among macrolide-resistant gonococcal strains
(37,233). Specific mutations of the macrolide target, 23S rRNA,
can also result in both low-level resistance (C2611T mutation)
(40) and high-level resistance (A2059G mutation) (37–39,42)to
erythromycin and azithromycin. The MICs of macrolides against
these resistant gonococcal isolates depend on how many of the
four 23S rRNA gene alleles contain the specific mutation. For
example, A2059G mutations in three or all four of the 23S rRNA
gene alleles result in high-level azithromycin resistance (MICs of
256 g/ml and up to 4,096 g/ml) (42), while strains with only
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596 cmr.asm.org Clinical Microbiology Reviews
one A2059G mutant allele can have an MIC of azithromycin sim-
ilar to that for wild-type strains. Strains with a single A2059G
mutation, while susceptible to azithromycin, quickly develop
high-level azithromycin resistance (MICs of 256 g/ml) upon
serial passage with subinhibitory concentrations of macrolides
(37).
Gonococcal resistance to macrolides because of overexpressed
efflux systems, particularly the MtrCDE efflux pump (234–240),
but also the MacAB (241) and mef-encoded (41,232,242) efflux
pumps, can affect the MICs of macrolides; this issue is discussed
below.
Cephalosporin Resistance
Cephalosporins, like other -lactam antimicrobials, inhibit the
cross-links of peptidoglycan within the bacterial cell wall by bind-
ing of the -lactam ring to PBPs (transpeptidases), which results
in bactericidal activity. Cephalosporin resistance in gonococci is
due primarily to mutations that modify the target proteins
(PBPs), but also to an increased efflux and decreased influx of
cephalosporin.
Like the case for chromosomally mediated penicillin resistance,
the primary ESC resistance determinants in N. gonorrhoeae are
specific alterations of the penA gene encoding PBP2, which is also
the main lethal target for cephalosporins (2–5,199,204). How-
ever, in contrast to the penA-Asp345a gene found in penicillin-
resistant strains, the penA gene from intermediate-level or fully
ESC-resistant strains is most frequently a mosaic gene that con-
tains up to 60 to 70 amino acid alterations, and notably, these
mosaic penA alleles do not contain the Asp345a insertion (3,5,
243). These mosaic penA alleles are considered to have emerged by
DNA transformation followed by recombination with partial
penA genes, particularly those from commensal Neisseria species
commonly residing in the oropharynx, such as Neisseria perflava,
Neisseria sicca,Neisseria polysaccharea,Neisseria cinerea, and/or
Neisseria flavescens (5,244–246). This in vivo intrageneric hori-
zontal transfer of entire or partial penA genes most likely occurred
during pharyngeal gonococcal infections (5,19,163,164). The
acquisition of a penA mosaic allele appears to increase the MICs of
cefixime more than those of ceftriaxone (3,4,199,204), which
might be caused by structure-function relationships due to the
longer C-3 side chain of the cephem skeleton of ceftriaxone (5,
243,246–248).
In gonococcal isolates with decreased susceptibility or resis-
tance to cefixime, three mutations in the mosica penA allele, re-
sulting in the amino acid alterations G545S, I312M, and V316T in
PBP2, were early suggested as important for the decreased ESC
susceptibility, particularly to cefixime (246,248). However, while
reversion of these three amino acids in PBP2 of a strain with de-
creased susceptibility or resistance to ESCs to those in wild-type
PBP2 dramatically decreased ESC resistance (MICs of ceftriaxone
and cefixime decreased 16- and 25-fold, respectively), introduc-
tion of only these three amino acids into a wild-type PBP2 se-
quence had little to no effect on resistance (MICs of ceftriaxone
and cefixime increased only 2- to 3-fold). Accordingly, these three
mutations increase resistance only in the presence of additional
mutations in the mosaic penA alleles that have limited effect on the
ESC MICs on their own, i.e., using a mechanism of epistasis (5,
204). These additional mutations might act as “compensatory” or
“stabilizing” mutations to restore transpeptidase activity essential
for the viability of the gonococcal strains. In the crystal structure
of PBP2, all three mutations are located near the -lactam active
site, with two residing on the same -helix as the serine nucleo-
phile, Ser310, of the SxxK active site motif. The G545S mutation is
present at the beginning of the 11 helix. The G545 and G546
main chain amides can bind (by hydrogen bonding) the side chain
hydroxyls of Thr498 and Thr500, respectively, located within the
KTG(T) active site motif. The main chain amide of Thr500 likely
forms the oxyanion hole that stabilizes the transition state. Ac-
cordingly, the G545S mutation might decrease acylation by inter-
fering with the structure of the transition state/tetrahedral inter-
mediate. Optionally, the Thr498 and Thr500 hydroxyl side chains
might interact with the carboxylate of the -lactam in the covalent
complex, and the acylation might be decreased by changes of these
contacts (204). Ile312 and Val316 are situated opposite Ser310 and
Lys313 in the SxxK active site motif on the 2 helix, and they pack
into a hydrophobic pocket. These interactions may be disrupted
and the location of the SxxK active site motif altered by mutations
to larger (I312M) or more hydrophilic (V316T) side chains, re-
sulting in lowered acylation (204). Furthermore, the N512Y alter-
ation in mosaic PBP2 has also been shown to be involved in
decreasing the susceptibility to ESCs, without affecting the suscep-
tibility to penicillin (204). This amino acid residue is located rel-
atively distant from the active site, on the same disordered 3-4
loop that harbors mutations associated with penicillin resistance,
and its reversion in mosaic PBP2 of a gonococcal strain with de-
creased ESC susceptibility to the wild type decreased the MICs of
ceftriaxone and cefixime 2-fold. Possibly, the N512Y alteration
perturbs the architecture of the KTG active site motif of the 3
strand (204). Finally, introduction of an A501V mutation, which
has been found mainly in gonococcal isolates with decreased ESC
susceptibility that lack a mosaic penA gene (246,249–251), into a
mosaic PBP2 increased the MICs of ceftriaxone and cefixime 2- to
4-fold, i.e., resulted in in vitro resistance (204), which further em-
phasizes the importance of the 3-4 loop of PBP2 for ESC resis-
tance.
Some additional PBP2 alterations, such as the G542S, P551S,
and P551L mutations, have also been associated statistically with
elevated MICs of ceftriaxone in gonococci (252). However, their
effects on the ceftriaxone MIC have not yet been proven with, e.g.,
site-directed penA mutations in isogenic strain backgrounds.
In regard to N. gonorrhoeae strains displaying high-level resis-
tance to all ESCs, the new mosaic penA allele in the first reported
XDR strain (from Kyoto, Japan), showing high-level resistance to
ceftriaxone and cefixime, contained 12 amino acid changes com-
pared to mosaic penA allele X, which was associated with most of
the early cefixime resistance and treatment failures in Japan (3).
Recently, it was verified that three of these mutations (A311V,
V316P, and T483S) resulted in the significant increases in the
MICs of ceftriaxone and cefixime (253). Ala311 and Val316 are
located on the same 2 helix of PBP2 as the Ser310 nucleophile.
Alterations in the hydrophobic packing of 2 caused by the A311V
and V316P mutations might modify the dynamics of transition
state formation and result in decreased ESC acylation. Further-
more, the bulky amino acid proline, known to promote helix
kinking, at position 316 might substantially influence the 2 helix
structure. The T483S mutation is highly conservative, but the loss
of the methyl group of Thr can have a substantial impact on ESC
acylation. Thr483 is located on a loop preceding the 3 strand that
comprises the KTG motif and is situated near the active site. Thus,
the T483S alteration may disturb the interaction with ESCs that
Antimicrobial Resistance Expressed by N. gonorrhoeae
July 2014 Volume 27 Number 3 cmr.asm.org 597
can increase the energy for activating the formation of the transi-
tion state and, accordingly, result in a lowered acylation rate,
and/or Thr483 might be important for binding ESCs, and the
T483S mutation increases binding and accordingly decreases the
second-order acylation (253). XDR and ESC-resistant gonococcal
strains have now been isolated in France (4) and Spain (2), and
both isolated strains belong to the multilocus sequence type
ST1901 and the N. gonorrhoeae multiantigen sequence type
ST1407 (254), which has been stated as a multidrug-resistant
clone accounting for a large proportion of the decreased suscep-
tibility and resistance to ESCs in many countries worldwide (5).
Both strains also contained a mosaic penA allele type XXXIV gene
(3) with an additional A501P alteration and displayed high-level
resistance to ceftriaxone and cefixime (2,4). Transformation ver-
ified that the new mosaic penA allele resulted in the ESC resistance.
It was also proposed that replacement of the methyl side chain of
Ala501, situated on the 3-4 loop, very close to the PBP2 KTG
active site motif (4,204), with the more bulky side chain of proline
(A501P) results in secondary structure changes and inhibits the
binding of ceftriaxone and cefixime to PBP2 by clashing with their
R1 substituents (4). In general, the full cause of ESC resistance in
all XDR gonococcal strains with high-level resistance to all ESCs
needs to be appropriately elucidated and verified, and this is in
progress. Site-directed mutagenesis experiments and crystal
structures of the altered forms of PBP2 in these strains are crucial
for detailing the molecular mechanisms underlying the reduced
acylation by ESCs, all mutations involved in influencing the MICs
of ESCs (mutations causing resistance and involved in epistasis),
and how the essential transpeptidase function is concomitantly
preserved. Furthermore, it is crucial to investigate the in vitro and,
particularly, in vivo biological fitness, e.g., in appropriate mouse
models, of these XDR gonococcal strains with high-level resis-
tance to all ESCs, and this is also in progress.
Finally, despite the fact that specific alteration of the lethal tar-
get PBP2 is the primary resistance mechanism, as for penicillin
(see above), an increased efflux and decreased influx of ESCs, due
to the mtrR resistance determinant (3–5,197–199) and penB re-
sistance determinants (3–5,199–201,249), respectively, result in
increased MICs of ESCs. Interestingly, both mtrR and penB have
greater effects on the MICs of ceftriaxone than on those of ce-
fixime, suggesting that cefixime is not an ideal substrate for either
the MtrCDE efflux pump or the PorB1b porin (199). The stepwise
transfer and interplay of the chromosomally mediated resistance
determinants have been elucidated for both penicillin (187) and
ESCs (199).
ponA (L421P) and pilQ (e.g., E666K) mutations (resistance de-
terminants for penicillin) do not seem to affect the MICs of ESCs
in presently circulating clinical gonococcal strains (5,187,199,
202). Most strains with decreased susceptibility or resistance to
ESCs contain the ponA1 allele. Nevertheless, this most likely re-
flects only that these strains evolved by horizontal transfer of mo-
saic penA alleles into preexisting chromosomally mediated peni-
cillin-resistant strains, which remain highly prevalent in the N.
gonorrhoeae population (5,199). pilQ2 (or any other pilQ loss-of-
function mutation) has never been reported in clinical isolates,
likely because it disrupts type IV pilus formation, and thus patho-
genic potential (5,203,255). Nevertheless, contributions to en-
hanced resistance of ponA and/or pilQ polymorphisms in future
ESC-resistant strains cannot be excluded. Finally, as with penicil-
lin resistance, the nontransformable “factor X” can influence the
MICs of ESCs (3–5,199,204).
Increased Efflux and Decreased Influx of Antimicrobials
Increased efflux. The capacity of cells to export drugs from their
interior was first described in the field of oncology, with the ob-
servation that the P-glycoprotein could export antitumor agents
(256). Since then, many bacterial efflux pumps have been de-
scribed, and they can be grouped into the following main catego-
ries based on their overall composition and the structures of their
transporter protein and the entire pump system: (i) the major
facilitator (MF) family, (ii) the small multidrug resistance (SMR)
family, (iii) the resistance-nodulation-cell division (RND) family,
(iv) the multidrug and toxic compound extrusion (MATE) fam-
ily, and (v) the ATP-binding cassette (ABC) family (89).
In gonococci, four efflux pump systems (MtrCDE, MacAB,
NorM, and FarAB) produced by all strains have been identified
(235,241,257,258). The MtrCDE, MacAB, NorM, and FarAB
systems belong to the RND, ABC, MATE, and MF families, re-
spectively, and the MtrCDE, MacAB, and NorM efflux pump sys-
tems have been shown to recognize antimicrobials previously or
currently recommended for gonorrhea treatment. Furthermore,
the mef-encoded efflux pump protein, which recognizes macro-
lides, has been identified in a few gonococcal strains, harbored on
a conjugative transposon (41,242,259). Based on decreases in
MICs of antimicrobials that result from insertional inactivation of
the gene encoding the cytoplasmic membrane transporter com-
ponent of the relevant pump, it has been shown that from the
periplasmic space, the MtrCDE efflux pump can export structur-
ally diverse hydrophobic antimicrobials, such as macrolides,
-lactam antimicrobials such as penicillin and ESCs, ciprofloxa-
cin, and tetracycline (198,234,235,237–240,260). The NorM
efflux pump exports fluoroquinolones (258), while the MacAB
efflux pump can export macrolides, and its loss has been linked to
increased susceptibility of gonococci to penicillin G and ESCs
(241,260).
The MtrCDE and FarAB efflux pumps also export host-derived
antimicrobials, including cationic antimicrobial peptides (261)
and long-chain fatty acids (257). Possession of an active MtrCDE
efflux pump is critical for gonococcal survival during experimen-
tal infection of the lower genital tract of female mice (262), and its
capacity to export host antimicrobials has been suggested to be
important for virulence (263) and gonococcal fitness in this
mouse infection model (171,172).
The MtrCDE pump is the efflux system that is studied most in
regard to gonococcal AMR. Expression of the mtrCDE efflux
pump operon is under the negative and positive control of both
cis- and trans-acting factors (Fig. 2). Gonococcal strains showing
intermediate-level resistance to substrates (hydrophobic drugs,
dyes, detergents, and host-derived antimicrobials [cationic pep-
tides and progesterone]) of the MtrCDE efflux pump (89) typi-
cally have missense mutations in a DNA-binding-domain coding
region of the mtrR gene (commonly a G45D mutation in the helix-
turn-helix [HTH] domain of amino acid residues 32 to 53 [264]),
which encodes the MtrR repressor that binds to the mtrCDE pro-
moter (265). However, strains expressing high-level resistance
have mutations (most frequently a single nucleotide [A] deletion
in the 13-bp inverted repeat sequence between the 10 and 35
hexamer sequences) in the overlapping mtrR promoter (237,239)
or, substantially more rarely, have a C-to-T transition 120 bp up-
Unemo and Shafer
598 cmr.asm.org Clinical Microbiology Reviews
stream of the mtrC translational start codon that generates a con-
sensus 10 hexamer sequence (TATAAT) and a novel promoter
for high-level transcription of mtrCDE outside the control of
DNA-binding proteins, such as MtrR (Fig. 2), which modulates
expression from the wild-type promoter (172,198). Other rare
alterations that increase expression of the MtrCDE efflux pump
have been described, e.g., a 153-bp Correia element (CE) (266)
insertion sequence located between the mtrR/mtrC promoter and
the mtrC gene (267,268). The emergence of this alteration sug-
gests that either a gonococcal CE element located elsewhere on the
chromosome was repositioned to the mtr locus or meningococcal
CE DNA sequences containing flanking mtr DNA were imported
and recombined at this site.
In regard to the MacAB and NorM efflux pump systems, mu-
tations upstream of the macAB and norM loci have been shown to
modulate levels of gonococcal susceptibility to antimicrobials by
altering gene expression (241,258). The macA and macB genes in
N. gonorrhoeae are organized as an operon, and the start point of
macAB transcription is located 37 nucleotides upstream of the
translational start codon. A SNP in the putative 10 hexamer
sequence (TAGAAT ¡TATAAT) has been shown to increase
transcription of macAB and to result in an increased resistance to
macrolides (241). Similarly, a SNP in the 35 hexamer sequence
(CTGACG ¡TTGACG) in the gonococcal norM promoter can
enhance transcription of norM, resulting in decreased susceptibil-
ity to norfloxacin and ciprofloxacin (258). Resistance levels could
be increased further by a second mutation that mapped to the
ribosome-binding site (TGAA ¡TGGA). While the levels of re-
sistance were insufficient to provide clinical resistance by them-
selves, they could be significant in strains expressing a level of
ciprofloxacin resistance near the MIC breakpoint.
Decreased influx. The Gram-negative outer membrane is an
important permeability barrier to different compounds, including
antimicrobials (269). Certain antimicrobials, such as penicillin,
tetracycline, and ESCs, diffuse into the periplasmic space of
Gram-negative bacteria through outer membrane channels
formed by porin proteins. A gonococcal strain produces one of
two mutually exclusive forms of porin, namely, PorB1a and
PorB1b (previously named major outer membrane protein, pro-
tein I, or Por), which are universally present as trimeric pore-
forming transmembrane porins (270). Strains expressing PorB1a
were early shown to be slightly more susceptible to penicillin and
tetracycline than strains expressing PorB1b (188,201,271). More-
over, amino acid alterations in loop 3 of PorB1b, which folds into
the barrel of the porin and constricts the pore, confer decreased
susceptibility of gonococci to penicillin, tetracycline, and ESCs
(199,201,202,271). Interestingly, penicillin and ceftriaxone are
more affected than cefixime, suggesting that either cefixime does
not readily diffuse into the periplasm through PorB1b or such
diffusion is not altered by the penB determinant. The net charge of
cefixime (2) also differs from the net charge of penicillin (1)
and ceftriaxone (1), which may affect the permeation properties
(202). The most-studied mutations (collectively named penB),
which result in amino acid replacements at position 120 alone
(G120K) or positions 120 and 121 (G120D/A121D), decrease an-
timicrobial entry (201). Interestingly, the phenotypic impact of
penB is only apparent in an mtrR mutant strain that overexpresses
the MtrCDE efflux pump (200,272).
PilQ is a doughnut-like multimeric secretin in the outer mem-
brane, composed of 12 identical 75-kDa subunits, that is used by
gonococci to secrete the growing pilus molecule (273,274). PilQ
also appears to be involved in controlling the influx of antimicro-
bials by gonococci, as specific mutations in pilQ have a significant
impact on levels of gonococcal susceptibility to structurally dis-
tinct antimicrobials (202,275). A spontaneously arising missense
mutation (penC) that decreased gonococcal susceptibility to pen-
icillin was earlier mapped to pilQ (202). Interestingly, penC
(E666K mutant; later renamed the pilQ2 mutation) had no effect
on the MICs of penicillins (or tetracycline and ciprofloxacin) in
the absence of the mtrR and penB resistance determinants, sug-
gesting that the rate of antimicrobial influx through the PilQ com-
plex in cells lacking mtrR and penB is only a small fraction of the
rate through porins (202). Only when mtrR and penB mutations,
which together substantially decrease antimicrobial levels in the
periplasm, are present is the influx through PilQ a significant pro-
portion of the total antimicrobial influx. Although PilQ has a role
in permeation of antimicrobials, it is unlikely that pilQ2-like mu-
tations or pilQ deletions have any role in clinical resistance, since
these mutations also decrease the stability of the PilQ dodecamer
and disrupt the proper piliation that is critical for the pathogenesis
of gonococci (203). In accordance with this, sequencing of pilQ
genes from a collection of geographically and temporally diverse
isolates (n63) with a range of ESC susceptibilities revealed nine
different PilQ amino acid sequences, but none of these showed a
significant association with increased MICs of ESCs (255).
ANTIMICROBIAL RESISTANCE AND GONOCOCCAL FITNESS
Bacterial strains with resistance to an antimicrobial have an ad-
vantage both in vitro and in vivo over antimicrobial-susceptible
FIG 2 Trans- and cis-acting regulatory elements that control expression of the
mtrCDE efflux pump operon in Neisseria gonorrhoeae.Trans-acting elements
behaving as repressors or genes that encode them are shown as barred lines
(), while those encoding activators are shown by arrows. The promoters
responsible for transcription of mtrR and mtrCDE are shown with their respec-
tive 10 and 35 hexamer sequences (see bars over hexamers). Note that
mtrR and mtrCDE are transcriptionally divergent, and only the mtrCDE cod-
ing strand is shown. The position of the 13-bp inverted repeat sequence (I.R.;
AAAAAGACTTTTT) between the 10 and 35 hexamers of the mtrR pro-
moter is shown within the 14 nucleotides in the dotted box, and a T nucleotide
that is frequently deleted in strains that overexpress mtrCDE and exhibit a high
level of resistance to pump substrates is shown in bold (329). The position of
the new 10 hexamer sequence generated by a point mutation (C to T) that
acts as a new promoter (mtr
120
) for mtrCDE transcription (198) is shown.
MtrR repression of mtrCDE is due to binding of two homodimers to the
mtrCDE promoter, as shown by the barred line that extends to the region
shown in the nucleotide sequence. MtrA binds upstream of the mtrCDE pro-
moter (265). (Adapted from reference 348 with permission.)
Antimicrobial Resistance Expressed by N. gonorrhoeae
July 2014 Volume 27 Number 3 cmr.asm.org 599
strains in the presence of the given antimicrobial; nevertheless,
they are commonly less fit in the absence of the antimicrobial
(276–278). However, compensatory/stabilizing/repairing muta-
tions that restore fitness while retaining the AMR occur frequently
in vitro, and likely also in vivo. Interestingly, in gonococci, AMR
mechanisms do not necessarily cause significantly lower biological
fitness, which results in the persistence of AMR and MDR/XDR
strains even in the absence of obvious antimicrobial pressure (the
antimicrobial is not used in the gonorrhea treatment anymore).
Nevertheless, selection due to the general antimicrobial pressure
in society remains. In fact, some AMR determinants (e.g., mtrR
and gyrA mutations) appear to even enhance the biological fitness
of at least some gonococcal strains. Consequently, many years
after penicillin, tetracycline, and fluoroquinolones were aban-
doned from the recommended treatment of gonorrhea, resistant
strains continue to represent a significant percentage of isolates
globally (5,8,19,81).
AMR and AMR determinants enhancing the fitness of bacteria
in vivo are exceedingly rare, but derepression of the gonococcal
mtrCDE efflux pump operon is one example. In a female mouse
(BALB/c) model of lower genital tract infections, null mutations
in mtrD or mtrE, resulting in loss of the MtrCDE efflux pump,
decreased the capacity of gonococcal strains to cause a sustained
(12-day) infection (262). This was likely caused by the inability of
the strain to export host-derived hydrophobic agents (e.g., anti-
microbial peptides and/or progesterone) by the MtrCDE efflux
pump (235,261). Furthermore, an mtrR null mutation that re-
sulted in loss of MtrR, a moderate overexpression of mtrCDE, and
enhanced AMR greatly increased the gonococcal fitness in vivo.In
contrast, loss of MtrA, which would abrogate the inducible over-
expression of the efflux pump (Fig. 2), decreased fitness in vivo.
However, this decrease in fitness could be reversed by compensa-
tory mutations that mapped to mtrR (171). Interestingly, clini-
cally isolated mutations in the mtr locus differ in the degree to
which they derepress the mtrCDE operon, and the resultant gra-
dient in erythromycin MICs parallels the degrees of resistance to
host-derived substrates and in vivo fitness conferred by these
mutations (172). It is important to remember that other MtrR-
and/or MtrA-regulated genes may also be important for the al-
tered fitness. In addition to regulating mtrCDE, MtrR can posi-
tively or negatively regulate 70 other genes in the gonococcal
genome, including glnA,glnE,ponA,pilMNOPQ, and rpoH (197,
279,280). There is also suggestive clinical evidence that the
MtrCDE efflux pump enhances the ability of gonococci to survive
during human infection. For instance, gonococcal mtrR mutants
have for decades been isolated frequently from cases of rectal gon-
orrhea (281–283), presumably because this environment is rich in
hydrophobic agents, such as long-chain fatty acids and bile salts.
The MtrCDE efflux pump probably also helps gonococci to evade
innate immune responses involving antimicrobial peptides, since
the pump can recognize the human cathelicidin LL-37 (261).
It was also recently shown that S91F and D95N mutations in
gyrA, resulting in intermediate resistance to ciprofloxacin, en-
hanced gonococcal fitness in vivo as assessed by competition with
the parental (wild type or mtrR negative) strain in the lower genital
tract of female mice (170). Interestingly, this fitness benefit was
not observed when the strains were cultured competitively in
vitro, and in fact, strains with both gyrA and mtrR mutations were
attenuated for growth relative to the parent strain. Addition of a
D86N mutation in parC, resulting in high-level ciprofloxacin re-
sistance, caused a substantial fitness cost in vivo. Nevertheless,
compensatory mutations that increased fitness while maintaining
ciprofloxacin resistance were selected in some mice. Subsequent
studies have shown that gyrA together with parC mutations in
other strain backgrounds have a fitness benefit similar to that con-
ferred by gyrA mutations alone, which suggests that ciprofloxacin-
resistant strains may spread even without acquiring compensatory
mutations.
ENVIRONMENTAL CONDITIONS AND GONOCOCCAL
RESISTANCE TO ANTIMICROBIALS
N. gonorrhoeae can respond to stresses imposed by its local envi-
ronment and alter the expression of genes involved in metabolism
and cell envelope structure. With respect to AMR, Rouquette et al.
(284) showed that expression of the mtrCDE-encoded efflux
pump is enhanced when gonococci are grown in the presence of
sublethal levels of pump substrates. This induction required the
presence of the MtrA protein (265,284), a member of the AraC
family of transcriptional activators (Fig. 2). In addition, expres-
sion of mtrCDE is linked to the availability of free iron (285). Such
iron-mediated regulation of mtrCDE involves the capacity of the
MpeR regulator to transcriptionally repress mtrR, encoding the
master repressor of mtrCDE, as well as mtrF (286)(Fig. 2). In turn,
mpeR expression is repressed by Fur in the context of iron (Fig. 2).
Thus, under conditions of low iron, mtrCDE expression could be
enhanced, allowing gonococci to increase their resistance to sub-
strates of the MtrCDE efflux pump, including conventional anti-
microbials and host-derived antimicrobials. Based on the work of
Warner et al. (171), resistance to the latter would increase the
fitness of gonococci during infection. It is important that this
model suggests that during infection, gonococcal resistance to an-
timicrobials recognized by this efflux pump may be elevated com-
pared to the resistance (in regard to MIC values) obtained under
laboratory conditions, which typically employ media rich in iron.
Interestingly, MpeR also transcriptionally activates expression of
fetA, which encodes a receptor for enterobactin-like siderophores
produced by other bacteria (287). Recent work (288) has shown
that both fetA and mpeR are upregulated during infection of hu-
man macrophages, suggesting that iron limitation in phagocytes
may enhance bacterial metabolism and AMR. Taken together,
conditions of iron limitation may support the ability of gonococci
to resist antimicrobials and proliferate during infection.
Gonococci also use host-derived compounds to resist antimi-
crobials. For instance, physiologic levels of polyamines (spermine
and spermidine) found in the male genital tract can coat the sur-
faces of bacteria, making them more resistant to complement-
mediated killing by normal human serum and cationic antimicro-
bial peptides (289). The importance of gonococcal resistance to
cationic antimicrobial peptides mediated by phosphoethano-
lamine (PEA) decoration of lipid A during infection was recently
documented (290). In this work, loss of the gene (lptA) encoding
the enzyme that adds PEA to the 4=position of lipid A decreased
gonococcal fitness during experimental genital tract infections of
female mice and human male volunteers.
Gonococci, like other bacteria (291), have the ability to form
biofilms under laboratory conditions, and this community life-
style also likely exists during infection (292,293). Bacteria grow-
ing in biofilms are often more resistant to antimicrobials than
their planktonic counterparts, and this property should be con-
sidered in evaluating the possible efficacy of newly developed an-
Unemo and Shafer
600 cmr.asm.org Clinical Microbiology Reviews
timicrobials to treat gonorrhea. Interestingly, recent work by Goy-
tia et al. (294) showed that gonococcal biofilm formation can be
blocked by the presence of spermine and spermidine, an observa-
tion that should be considered in testing the efficacy of new anti-
microbials.
INTERNATIONAL RESPONSES TO RESISTANCE EVOLUTION
AND POSSIBLE EMERGENCE OF UNTREATABLE GONORRHEA
The evolution of AMR in gonococci, and particularly the emer-
gence of resistance to ceftriaxone, is of great concern internation-
ally. As a response to this serious situation, recommendations to
use dual-antimicrobial treatment regimens for uncomplicated
anogenital and pharyngeal gonorrhea were introduced in all of
Europe (17), the United States (15), the United Kingdom (16),
and some regions within countries (e.g., Ontario, Canada [295]).
These treatment guidelines recommend intramuscular ceftriax-
one (250 mg [15] or 500 mg [16,17,295], in a single dose) together
with either oral azithromycin (1 g [15,16,295]or2g[17]) or
doxycycline (100 mg twice daily for 7 days) (15), which will also
eradicate concomitant C. trachomatis infections. These dual-anti-
microbial treatment regimens currently appear to be highly effec-
tive and can be recommended.
Furthermore, the WHO has published a global action plan (13,
14), and the U.S. CDC (11) and the European Centre for Disease
Prevention and Control (ECDC) (12) have published regional
response plans, to control and mitigate the spread of multidrug-
resistant N. gonorrhoeae. A key component of these action/re-
sponse plans is to substantially enhance the quality-assured sur-
veillance of gonococcal AMR and gonorrhea treatment failures
(using recommended treatments), locally, nationally, and inter-
nationally. The WHO Global GASP was established in the early
1990s but was relaunched in 2009 (13). The WHO GASP aims to
act in close liaison with other existing GASPs; for example, in the
European Union/European Economic Area (EU/EEA), the ECDC
is responsible for the regional Euro-GASP (105,106,130,296),
and national GASPs exist in the United States (297;http://www
.cdc.gov/std/gisp), the United Kingdom (298;http://www.hpa
.org.uk/Publications/InfectiousDiseases/HIVAndSTIs/GRASP
Reports/), and several additional countries. This enhanced gono-
coccal AMR surveillance should monitor trends in resistance (in-
cluding regional differences), identify newly emerging AMR, and
inform treatment recommendations in a timely manner. Worry-
ingly, longitudinal quality-assured GASPs have been incomplete
in some regions (such as Latin America and the Caribbean [99,
129]) or have been sporadic or completely lacking in large parts of
the world, including Eastern Europe, Central Asia, and Africa
(104,106,152). The latter regions also suffer from a high burden
of gonorrhea, creating the prerequisites for rapid emergence and
spread of gonococcal AMR. Accordingly, it is crucial to establish
and maintain gonococcal culture and AMR surveillance in these
regions and others worldwide. This requires significant political
will and funding as well as an investment in laboratory infrastruc-
ture and staff training. However, all the action/response plans also
strongly emphasize the importance of more holistic actions. These
include, for example, to also combat the global burden of gonor-
rhea and substantially improve the early prevention, diagnosis,
contact tracing, treatment, and epidemiological surveillance of
gonorrhea cases. These efforts need to be combined with wider
strategies for general antimicrobial control (guidelines for appro-
priate use, selection, supplies, quality, etc.) and an increased
awareness among microbiologists, epidemiologists, and clinicians
and on political levels; the latter is especially important for financ-
ing efforts in gonorrhea control. In general, there is an urgent need
for an enhanced focus on reducing gonorrhea burdens in high-
risk, frequently transmitting populations (such as CSWs and
MSM), as well as appropriate diagnosis and treatment of pharyn-
geal gonorrhea, which is harder to eradicate and is an asymptom-
atic reservoir for gonorrhea-causing bacteria and emergence of
AMR. Finally, many crucial research needs should be a priority
and should be stressed, including substantially intensifying re-
search efforts to identify or develop alternative therapeutic strat-
egies and, particularly, compounds for treatment of gonorrhea
(14).
FUTURE PERSPECTIVES FOR TREATMENT
The dual-antimicrobial treatment regimens with ceftriaxone and
azithromycin introduced in the United States (15) and Europe
(17) appear to currently be highly effective and should be consid-
ered in all settings where comprehensive, quality-assured local
AMR surveillance data are lacking or not clearly supporting any
other treatment regimen (298,299). However, the susceptibility to
ceftriaxone has been decreasing globally in recent years, resistance
to azithromycin is prevalent in many settings and emerges rapidly
in settings where this drug is frequently used, and gonococcal
strains with decreased susceptibility or resistance to ceftriaxone
and concomitant azithromycin resistance are already circulating
globally. Gonococcal strains with high-level resistance to azithro-
mycin (MICs of 256 g/ml) are also reported from an increas-
ing number of countries (37–39,42,132,133). Furthermore, dual-
antimicrobial therapy might not be affordable in lower-resource
settings, many of which suffer from the highest gonorrhea bur-
dens, and consequently may not significantly mitigate AMR emer-
gence and spread globally (5,8,299). This suggests that the cur-
rently recommended dual-antimicrobial regimens will not be
effective long-term solutions. From a global public health per-
spective, novel antimicrobials or other therapeutic compounds
for effective monotherapy, or at least for inclusion in a new dual-
therapy regimen, are essential. A randomized clinical multicenter
trial was recently conducted to evaluate gentamicin (240 mg given
once intramuscularly) plus azithromycin (2 g given once orally)
and gemifloxacin (320 mg given once orally) plus azithromycin (2
g given once orally) as potential alternative treatment options for
uncomplicated gonorrhea, that is, as a potential salvage therapy if
widespread ceftriaxone resistance emerges. Microbiological cure
was accomplished by 100% of the gentamicin-azithromycin pa-
tients and by 99.5% of gemifloxacin-azithromycin patients. Un-
fortunately, adverse events were relatively frequent in both dual-
antimicrobial arms (300).
The parenteral aminoglycoside gentamicin has been used as a
first-line treatment for 20 years in Malawi, without any obvious in
vitro resistance (301,302), and in Europe the in vitro susceptibility
is also high (303). Nevertheless, in Malawi, gentamicin has
mainly not been used for gentamicin monotherapy but instead
for syndromic management of urogenital infections, together
with doxycycline, and no data regarding treatment efficacy
against pharyngeal or anorectal gonorrhea exist. Furthermore,
correlates between MICs of gentamicin, pharmacokinetic/phar-
macodynamic parameters, and treatment outcomes and, conse-
quently, evidence-based microbiological resistance breakpoints
are lacking. Finally, single-dose gentamicin treatment had a
Antimicrobial Resistance Expressed by N. gonorrhoeae
July 2014 Volume 27 Number 3 cmr.asm.org 601
pooled cure rate of only 91.5% in a recent meta-analysis (304).
The novel oral fluoroketolide solithromycin was recently shown
to have high in vitro activity against gonococci, including ESC-
resistant XDR and MDR isolates (305). A recent minor phase 2
single-center, open-label study also showed that a single oral dose
of solithromycin (1.2 g) successfully treated all 22 evaluable pa-
tients with uncomplicated gonococcal infection (306). Neverthe-
less, gonococcal isolates with high-level resistance to azithromycin
(MICs of 256 g/ml) have solithromycin MICs of 4 to 32 g/ml,
which indicates resistance (305). The in vitro activity of ertapenem
(parenteral 1--methyl-carbapenem) against gonococci, includ-
ing MDR and XDR isolates, is also high (307). However, ESC
resistance determinants, i.e., mosaic penA alleles, mtrR, and penB,
also increase the ertapenem MIC (307). Accordingly, future re-
modeling of PBP2, further decreasing acylation by ertapenem and
working synergistically with the existing resistance determinants,
appears likely. Gentamicin, solithromycin, and possibly ertap-
enem might be options for future treatment of gonorrhea, but
probably not for first-line empirical monotherapy and instead for
salvage therapy for ceftriaxone-resistant cases and/or as one of the
antimicrobials in a dual-antimicrobial treatment regimen.
Regarding the pipeline of derivatives of previously developed
antimicrobials, some novel drugs have been evaluated in vitro
against N. gonorrhoeae isolates. The broad-spectrum parenteral
glycylcycline tigecycline, a tetracycline derivative, showed potent
in vitro activity against gonococci, including tetracycline-resistant
strains (308,309). Worryingly, tigecycline is mostly eliminated
through the bile, and a limited proportion of the given antimicro-
bial is excreted unchanged in the urine, which calls into question
the use of tigecycline to treat urinary tract infections (310–312).
Another novel and fully synthetic tetracycline analog (fluorocy-
cline), eravacycline (TP-434), was recently shown to have a high in
vitro activity against tetracycline-, penicillin-, and ciprofloxacin-
resistant gonococcal isolates (313). The semisynthetic lipoglyco-
peptide dalbavancin recently showed potent activity against a
small number of gonococcal isolates (314). Two new broad-spec-
trum parenteral 2-acyl carbapenems, SM-295291 and SM-
369926, showed high in vitro antimicrobial activities against gono-
cocci, including ciprofloxacin-resistant isolates (315). Two novel
broad-spectrum fluoroquinolones, avarofloxacin (JNJ-Q2) (316)
and delafloxacin (317), also displayed high in vitro activities
against gonococcal isolates, including ciprofloxacin-resistant iso-
lates. A phase 3 clinical trial has also been designed to compare
delafloxacin (2 450-mg tablets administered once) to ceftriaxone
(250 mg given once intramuscularly) for treatment of uncompli-
cated gonorrhea (http://clinicaltrials.gov/show/NCT02015637).
Nevertheless, it is important to emphasize that N. gonorrhoeae
has developed resistance to all antimicrobials introduced for first-
line therapy during the last 70 to 80 years, and for more sustain-
able future treatment, it might be essential to “think outside the
box.” Thus, it is imperative to focus not only on derivatives of
previously developed antimicrobials but also on the development
and investigation of new targets, compounds, and approaches for
treatment. This could be a novel single target; however, ideally,
multiple targets, compounds, and/or approaches to also suppress
resistance development will retain the antimicrobials for longer
use. Notably, several antimicrobials or other compounds, using
new targets or mechanisms, were recently developed, and several
have shown high in vitro activities against gonococcal isolates.
These include novel inhibitors of protein synthesis, e.g., pleuro-
mutilin BC-3781 (318,319) and the boron-containing inhibitor
AN3365 (320,321); novel inhibitors of bacterial topoisomerases
that target regions different from the fluoroquinolone-binding
sites (322,323), such as VT12-008911 (324) and AZD0914 (323,
325,326); FabI inhibitors, such as MUT056399 (327,328); non-
cytotoxic nanomaterials (329); inhibitors of efflux pumps, partic-
ularly coadministered with appropriate antimicrobials, that in-
crease the susceptibility to certain antimicrobials, the innate host
defense, and toxic metabolites (234,260,330); LpxC inhibitors
(331); molecules mimicking host defensins; host defense peptides,
such as LL-37 (multifunctional cathelicidin peptide) (332); and
IL-12 NanoCap, which is a biodegradable sustained-release for-
mulation of human interleukin 12 that aims to be a therapeutic
vaccine against N. gonorrhoeae (TherapyX Inc.). Several of these
novel antimicrobials or other types of compounds deserve further
attention for their potential use as future treatments for gonor-
rhea. For example, the activities of many of these have not been
examined against XDR or MDR gonococcal isolates, particularly
those with ESC resistance or high-level azithromycin resistance.
Furthermore, despite the fact that a breakthrough is lacking, as
part of “thinking outside the box,” it can be important to contin-
uously evaluate appropriate plant extracts for in vitro activity
against gonococci and, subsequently, for treatment of gonorrhea
(136,333–345). All novel potential treatment regimens for gon-
orrhea require up-to-date and comprehensive in vitro and, subse-
quently, in vivo evaluations, including appropriately designed,
randomized, and controlled clinical trials to evaluate parameters
such as efficacy, safety, toxicity, cost, optimal dose, and pharma-
cokinetic/pharmacodynamic data for genital and extragenital (es-
pecially pharyngeal) gonorrhea. Furthermore, knowledge regard-
ing current and future genetic resistance determinants (in vitro
selected and in vivo emerged) for these antimicrobials (both in
gonococci and in bystander organisms during treatment of gon-
orrhea), clear correlates between genetic and phenotypic labora-
tory parameters, and clinical treatment outcomes would be ex-
ceedingly valuable.
Additional knowledge regarding the structure and evolution of
bacterial targets for antimicrobials or those that participate in re-
sistance, prediction of the evolution of these targets and, accord-
ingly, emergence of resistance, and whether the current or soon-
to-emerge resistance mechanisms have a fitness cost or benefit will
provide new opportunities for development of effective and sus-
tainable antimicrobials. Genomic, transcriptomic, and proteomic
studies coupled with additional advances in drug chemistry and
high-throughput screening of compound libraries and insights
gained from physiological experiments will identify novel bacte-
rial targets and provide opportunities for rational design of new
drugs. High-throughput genome sequencing and other novel mo-
lecular technologies, combined with appropriate epidemiological
metadata and phylogenomic and phylogeographic analyses, will
also provide an enhanced understanding of the dynamics of the
national and international emergence, transmission, and evolu-
tion of antimicrobial-resistant gonococcal strains and might ad-
ditionally revolutionize molecular AMR testing for both gonococ-
cal isolates and NAAT-positive samples (346,347). It might even
be elucidated how, when, and where successfully transmitted
gonococcal strains and their possible AMR emerge, further evolve,
and spread (including mechanisms and time scales) in communi-
ties, nationally and internationally. This knowledge is imperative
to mitigate and, ideally, predict the emergence and spread of
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602 cmr.asm.org Clinical Microbiology Reviews