SURVEY OF OPHTHALMOLOGY VOLUME 49 • SUPPLEMENT 2 • MARCH 2004

Fluoroquinolones: Mechanism of Action, Classification,

and Development of Resistance
Joseph M. Blondeau, MSc, PhD, RSM (CCM), SM (AAM), FCCP

Department of Clinical Microbiology, Royal University Hospital and the Departments of Microbiology and Immunology
and Pathology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Abstract. The fluoroquinolones represent an evolving class of broad-spectrum antimicrobial agents

used in the prevention and treatment of a variety of ocular infections; however, resistance to currently
available agents in the class has been emerging among ocular pathogens. This article reviews the
mechanism of action of existing and new fluoroquinolones and discusses the structure–activity
relationship of the fluoroquinolones as it relates to the classification of these compounds. This article
also highlights the mechanism of resistance among common ocular pathogens and discusses the poten-
tial need for newer fluoroquinolones in ophthalmology. (Surv Ophthalmol 49(Suppl 2):S73–S78,
2004. 쑖 2004 Elsevier Inc. All rights reserved.)

Key words. antimicrobials • fluoroquinolones • mechanisms of action • ocular infections •

resistance topoisomerases

Ocular infections, both superficial and deep, such variety of Gram-positive organisms, including Strepto-
as conjunctivitis, corneal ulcers, and endophthal- coccal and Staphylococcal species; however, resistance
mitis, are caused by a diverse group of bacterial, is emerging among some of these organisms.10,13,32
viral, and fungal pathogens. Accordingly, the arma- This article briefly reviews the mechanism of action
mentarium of available antimicrobials used in the and classification of fluoroquinolones. This article
prevention and treatment of these infections in- also discusses the emergence of resistance to fluoro-
cludes antivirals, antifungals, and antibacterials. quinolones from a mechanistic standpoint and ad-
Common topical antibacterials used in the treatment dresses the potential role for newer agents in the
of ocular infectious diseases include sulfonamides, class.
aminoglycosides, polymyxin-based combinations,
and fluoroquinolones.22
The fluoroquinolones represent an expanding Mechanism of Action
class of broad-spectrum antibacterials which cover a Fluoroquinolones act by inhibiting two enzymes
host of Gram-negative and anaerobic species respon- involved in bacterial DNA synthesis, both of which
sible for ocular infections.14,39 These antibacterials are DNA topoisomerases that human cells lack and
have gained popularity in the ophthalmology field that are essential for bacterial DNA replication,
since they have been shown to be equivalent to combi- thereby enabling these agents to be both specific and
nation therapy in the treatment of many ocular infec- bactericidal.13,32 DNA topoisomerases are responsi-
tions.32 Fluoroquinolones are also effective against a ble for separating the strands of duplex bacterial

S73
쑖 2004 by Elsevier Inc. 0039-6257/04/$–see front matter
All rights reserved. doi:10.1016/j.survophthal.2004.01.005
S74 Surv Ophthalmol 49 (Suppl 2) March 2004 BLONDEAU

DNA, inserting another strand of DNA through the Older fluoroquinolones exhibit a relatively con-
break, and then resealing the originally separated sistent pattern with respect to specificity of enzyme
strands.34 inhibition in different types of bacteria. Although
Specifically, fluoroquinolones inhibit DNA gyrase some degree of overlap may exist, DNA gyrase tends
and topoisomerase IV. DNA gyrase introduces nega- to be the primary target for fluoroquinolones in Gram-
tive superhelical twists in the bacterial DNA double- negative organisms whereas topoisomerase IV is typi-
helix ahead of the replication fork, thereby catalyzing cally the primary target in Gram-positive bacteria.13,32
the separation of daughter chromosomes. This activ- This mechanistic specificity has important im-
ity is essential for initiation of DNA replication and plications in the development of resistance to older
allows for binding of initiation proteins. DNA gyrase fluoroquinolones and in the efficacy of newer fluoro-
is composed of two GyrA and two GyrB monomeric quinolones against resistant strains. Data from trials
subunits, which are encoded by the gyrA and gyrB in resistant mutant species suggest that newer, fourth-
genes, respectively.4,29,35 Topoisomerase IV is respon- generation fluoroquinolones (see Classification and
Structure-activity Relationships section) likely have a
sible for decatenation, that is, removing the inter-
dual-binding mechanism of action, inhibiting both
linking of daughter chromosomes, thereby allowing
DNA gyrase and topoisomerase IV, in Gram-positive
segregation into two daughter cells at the end of a
species.5,31 Although some have debated the dual-
round of replication.39 Topoisomerase IV is com-
activity of newer fluoroquinolones based on enzymic
posed of four homologous monomeric subunits, two and genetic studies,33 the general consensus is that
ParC (GrlA in Staphylococcus aureus) subunits and some newer agents, such as gatifloxacin and moxi-
two ParE (Grl B in S.aureus) subunits encoded by the floxacin, do possess dual-activity.8 Owing to the rarity
parC and parE genes, respectively.18 The structure and of double mutations (e.g., 10⫺14 for fluoroquinolones
function of DNA gyrase and topoisomerase IV are in Streptococcus pneumoniae), the preferential use of
outlined in Fig. 1. such agents could potentially limit the emergence
Fluoroquinolones interact with the enzyme-bound of fluoroquinolone resistance.33
DNA complex (i.e., DNA gyrase with bacterial DNA
or topoisomerase IV with bacterial DNA) to create Classification and Structure-activity
conformational changes that result in the inhibition Relationships
of normal enzyme activity. As a result, the new drug– A number of different classification systems have
enzyme–DNA complex blocks progression of the rep- been used in the literature to describe the evolution
lication fork, thereby inhibiting normal bacterial of the quinolone class of antibacterials,1–3,5,20,25,27 and
DNA synthesis and ultimately resulting in rapid bacte- this issue continues to be a source of debate. Here,
rial cell death.17,24 we outline a classification system that is designated
by various generations of quinolones, which correlate
with clinical utility. This system is focused on fluoro-
quinolones used in ocular infectious diseases, and
the structure-activity relationships of relevant com-
pounds are also presented.
Quinolones are synthetic molecules developed
through structural modification of the ‘4-quinolone’
skeleton (Fig. 2).27,32 The first quinolone developed

Fig. 1. Structure and principal function of DNA gyrase and Fig. 2. ‘4-quinolone’ skeleton (4-oxo-1, 4 dihydroquino-
topisomerase IV. (Reprinted from Hooper13 with permis- lone). (Reprinted from Smith et al32 with permission of
sion of Drug Reisitance Updates.) Drugs.)
FLUOROQUINOLONES S75

was nalidixic acid, which was found to have limited ac- Gram-positive activity associated with fluorination in
tivity against Gram-negative organisms. Clinical use this position, combined with solubility in ophthalmic
was limited to the management of uncomplicated solutions for selected third-generation compounds,
urinary tract infections. Structural modification of expanded the clinical utility of quinolones to the
this first-generation quinolone ensued, in an effort ophthalmology field.
to expand the spectrum of activity. The structural Norfloxacin was the first quinolone used in the
evolution of the quinolones is outlined in Fig. 3. management of ocular infectious diseases, intro-
Marginal structural modifications were initially duced for the treatment of bacterial conjunctivitis.
made in order to develop some of the early second- Norfloxacin demonstrated anti-pseudomonal activ-
generation compounds such as oxolinic acid and ity, activity against Gram-negative bacilli, as well as
cinoxacin. More substantial improvements in Gram- limited activity against susceptible Gram-positive bac-
negative coverage, including antipseudomonal activ- teria.15 Carbon for nitrogen substitution at the X
ity, were achieved with piperazine substitution at the position (attached to the R8 side-chain in Fig. 2)
R7 position, as with the development of pipemidic along with side-chain modifications at the R1 and
acid2 (Fig. 3). Although some degree of expanded R8 positions led to the development of clinically im-
antibacterial activity was achieved, clinical use of proved third-generation compounds. The addition
second-generation quinolones remained limited by of a cyclopropryl ring at the R1 position led to the
poor systemic bioavailability and the risk of renal development of ciprofloxacin, and the addition of a
toxicity.1,20 six-member (pyridobenzoxazine) ring between the
Third-generation quinolones were marked by fluo- R1 and R8 positions led to the development of ofloxa-
rination at the R6 position, giving rise to the classifica- cin.3 Both offered improved Gram-negative suscepti-
tion of future compounds as fluoroquinolones.3 The bility as well as expanded activity against frequently

Fig. 3. Structural evolution of fluoroquinolones. (Modified from Blondeau5 with permission of Expert Opinion on Investiga-
tive Drugs.)
S76 Surv Ophthalmol 49 (Suppl 2) March 2004 BLONDEAU

encountered Gram-positive organisms in ocular in- tend to confer lower levels of resistance than do
fections. Ofloxacin also exhibits activity against GyrA mutations.13
anaerobes such as Propionibacterium acnes.25 Levoflox- DNA gyrase mutations, both in the GyrA and GyrB
acin, created by isolation of the active enantiomer of subunits, also occur in Gram-positive bacteria; how-
ofloxacin, conferred further improved susceptibility ever, as DNA gyrase typically serves as a secondary
against Gram-positive bacteria, including susceptible target to topoisomerase IV in these bacterial species,
strains of Strep. pneumoniae and Strep. viridans.1,25 the relative importance of these mutations is question-
The expanded coverage of these newer third-genera- able. DNA gyrase appears to be the primary target for
tion fluoroquinolones made them preferred agents gatifloxacin and moxifloxacin in S.pneumoniae. Fur-
as first-line topical therapy for infections such as bac- thermore, in fluoroquinolone-resistant Gram-posi-
terial keratitis.23,28 tive bacteria, alterations in either subunit of DNA
Emerging resistance among ocular pathogens to gyrase have typically been found only in the presence
third-generation fluoroquinolones, particularly among of concomitant topoisomerase IV mutations.13,21
Gram-positive organisms, has led to the development Topoisomerase IV mutations have been most exten-
of newer agents.7,10,25 The addition of a methoxy side- sively studied in fluoroquinolone-resistant Gram-posi-
chain at the R8 position led to the development of tive bacteria, particularly in S.aureus and S.pneumoniae
fourth-generation compounds including gatifloxacin species. As with DNA gyrase, alterations may occur in
and moxifloxacin. Moxifloxacin also has a bulky bicy- either subunit, mutations in ParC (GrlA in S.aureus)
clic ring attached at the R7 position.1,5 Gatifloxacin occur more commonly than ParE (GrlB in S.
also carries a methyl group on the piperazinyl ring. aureus) mutations, and ParC mutations are believed
These modifications are believed to allow for the dual to play a more critical role in resistance develop-
mechanism of action in Gram-positive bacteria in ment.26 The ParC subunit has an analogous QRDR
addition to reducing efflux from the bacterial cell, domain and resistance is also believed to be mediated
thereby improving the spectrum of activity of these through reduced drug affinity.13 Topoisomerase IV
agents to include strains of Streptococcus and Staphylo- alterations have also been studied in Gram-negative
coccus species otherwise resistant to older generation bacteria and are believed to play a secondary role in
fluoroquinolones.9,25 The methoxy group is also be- development of resistance as ParC or ParE mutations
lieved to confer added anaerobic activity to the typically confer resistance only in the presence of
fourth-generation compounds.1 concomitant DNA gyrase mutations.6,19
In order for fluoroquinolones to access their cyto-
plasmic drug targets, these agents must cross the cell
Mechanism of Resistance wall and cytoplasmic membrane in all bacteria, as
Fluoroquinolone resistance develops through two well as the outer membrane of Gram-negative organ-
main mechanisms: alterations in the drug target en- isms. An alternative mechanism of bacterial resis-
zymes and alterations in access to the drug target tance has emerged through expression of multidrug
enzymes. Alterations in the drug target enzymes may resistant (MDR) membrane-associated efflux pumps,
be sub-classified as mutations in DNA gyrase and mu- which actively pump drug out of the bacterial cell.
tations in topoisomerase IV. These mutations differ MDR efflux pumps have been shown to reduce fluor-
among the bacterial types (i.e., Gram-negative vs. oquinolone activity and contribute to low-level resis-
Gram-positive) in terms of their relative impact on tance.13 Additionally, in Gram-negative organisms,
fluoroquinolone resistance development.13 decreased levels of outer membrane proteins respon-
Alterations in DNA gyrase occur most commonly sible for drug diffusion, such as general diffusion
in fluoroquinolone-resistant Gram-negative bacteria porins, have also been shown to reduce the accumula-
and may affect either the GyrA or GyrB subunits. Mu- tion of drug within the cytoplasm.11,12,30 This mech-
tations in the GyrA subunit tend to cluster in a region anism of reducing drug diffusion into the cell acts
known as the quinolone resistance-determining in concert with MDR efflux pumps to contribute to
region (QRDR). The QRDR represents the region bacterial resistance.13
of the gyrA gene encoding the portion of the GyrA While an overview on the mechanisms of resistance
subunit that is bound to DNA during enzyme activ- is provided, the relevance of the different resis-
ity.38 The most common mutations in this region are tance mutations and their impact on drug susceptibil-
believed to cause resistance through decreased drug ity is not discussed. Similarly, discussion regarding
affinity for the altered gyrase-DNA complex.36 GyrB various levels of susceptibility/resistance in reference
mutations occur less commonly than GyrA mutations. to serum susceptibility breakpoints and the rel-
They cluster in an analogous QRDR domain; how- evance of these breakpoints to infection and achiev-
ever, it is uncertain whether GyrB mutations in this able drug concentrations at various anatomical
region affect drug binding.37 Alterations in GyrB also locations is not discussed. Some of these issues have
FLUOROQUINOLONES S77

been previously discussed and readers are referred 4. Blondeau JM: Expanded activity of the new fluoroquinolones:
a review. Clin Therapeu 21:3–40, 1999
to Blondeau5 and Jacobs et al.16 Although these arti- 5. Blondeau JM: A review of clinical trials with fluoroquinolones
cles do not specifically address resistance, drug con- with an emphasis on new agents. Exp Opin on Investigational
centrations, and ocular infections, they nonetheless Drugs 9:383–413, 2000
6. Breines DM, Ouabdesselam S, Ng EY, et al: Quinolone resis-
discuss the issues of serum breakpoints being applied tance locus nfxD of Escherichia coli is a mutant allele of parE
for infection at all anatomical locations. gene encoding a subunit of topoisomerase IV. Antimicrob
Agents Chemother 41:175–9, 1997
7. Chaudry NA, Flynn HW, Murray TG, et al: Emerging ci-
Summary profloxacin-resistant Pseudomonas aeruginosa. Am J Ophthal-
mol 128:509–10, 1999
The fluoroquinolones represent a diverse class of 8. Fisher LM, Heaton VJ: Dual activity of fluoroquinolones
against Streptococcus pneumoniae. J Antimicrob Chemother 51:
bactericidal agents with multiple applications in 463–4, 2003
ocular infectious diseases. Their mechanism of 9. Fukuda H, Kishii R, Takei M, et al: Contributions of the 8-
action specifically targets bacterial DNA synthesis, and methoxy group of gatifloxacin to resistance selectivity, target
evolving generations of these compounds have led peference and antibacterial activity against Streptococcus pneu-
monaie. Antimicrob Agents Chemother 45:1649–53, 2001
to improved broad-spectrum coverage. Despite new 10. Goldstein MH, Kowalski RP, Gordon YJ: Emerging fluoroqui-
drug development, bacterial species have evolved in nolone resistance in bacterial keratitis. Ophthalmology 106:
a parallel manner to develop novel mechanisms of 1313–1318, 1999
11. Hirai K, Aoyana H, Irikura T, et al: Differences insusceptibility
bacterial resistance. to quinolones of outer membrane mutants of Salmonella typhi-
Topical fourth-generation fluoroquinolones gati- murium and Escherichia coli. Antimicrob Agents Chemother
floxacin and moxifloxacin have been approved re- 29:535–8, 1986
12. Hirai K, Suzue S, Irikura T, et al: Mutations producing resis-
cently by the U.S. Food and Drug Administration for tance to norfloxacin in Pseudomonas aeruginosa. Antimicrob
ocular indications. They represent the most advanced Agents Chemother 31:582–6, 1987
group of compounds within the class and offer a 13. Hooper DC: Mechanisms of fluoroquinolone resistance.
Drug Resist Updates 2:38–55, 1999
unique dual-binding mechanism of action in Gram- 14. Hooper DC, Wolfson JS: Fluoroquinolone antimicrobial
positive organisms, displaying activity against otherwise agents. N Engl J Med 324:384–94, 1991
resistant species. Although further clinical evidence of 15. Ito K, Hirai K, Inoue M, et al: In vitro antibacterial activity
of AM-715, a new nalidixic acid analog. Antimicrob Agents
their efficacy in prophylaxis and treatment of ocular Chemother 17:103–8, 1980
infections is required, there is a growing need for com- 16. Jacobs MR, Bajaksouzian S, Zilles A, et al: Susceptibilities of
pounds of this nature to combat emerging resistance. Streptococcus pneumoniae and Haemophilus influenzae to 10
oral antimicrobial agents based on pharmacodynamic param-
eters: 1997 U.S. Surveillance Study. Antimicrob Agents Che-
mother 43:1901–8, 1999
Method of Literature Search 17. Kampranis SC, Maxwell A: Conformational changes in DNA
A literature search for this article was performed gyrase revealed by limited proteolysis. J Biol Chem 273:
22606–14, 1998
based on medline database searches from 1966 to 18. Kato JI, Suzuki H, Ikeda H: Purification and characterization
present, using varying combinations of the search of DNA topoisomerase IV in Escherichia coli. J Biol Chem
terms ocular infections, fluoroquinolones, classification, 267:25676–84, 1993
19. Khodursky AB, Zechdiedrich EL, Cozzarelli NR: Topoisomer-
generations, mechanism of action, structure, activity, DNA ase IV is a target of quinolones in Escherichia coli. Proc Natl
gyrase, topoisomerase IV, and resistance. Relevant English Acad Sci USA 92:11801–5, 1995
journal articles and/or abstracts were selected for 20. King DE, Malone R, Lilley SH: New classification and update
on the quinolone antibiotics. Am Fam Physician 61:2741–
review. Articles cited in the references of journal arti- 8, 2000
cles were also selected, as appropriate. The publica- 21. Korten V, Huang WM, Murray BE: Analysis by PCR and direct
tions cited in this manuscript represent some of the DNA sequencing of gyrA mutations associated with fluoroqui-
nolone resistance in Enterococcus faecalis. Antimicrob Agents
best information that is currently available on the Chemother 38:2091–4, 1994
fluoroquinolones and I am unaware of a similar 22. Leeming JP: Treatment of ocular infections with topical anti-
collection of manuscripts that have been published bacterials. Clin Pharmacokinet 37:351–60, 1999
more recently that cover the same spectrum of mate- 23. Leibowitz HM: Clinical evaluation of ciprofloxacin 0.3% oph-
thalmic solution for treatment of bacterial keratitis. Am J
rial as covered in the references used. Ophthalmol 112(Suppl):34S–47S, 1992
24. Marlans KJ, Hiasa H: Mechanism of quinolone action—a
drug-induced structural pertubation of the DNA precedes
References strand cleavage by topoisomerase IV. J Biol Chem 272:9401–
9, 1997
1. Appelbaum PC, Hunter PA: The fluoroquinolone antibacteri- 25. Mather R, Karenchak LM, Romanowski EG, Kowalski RP:
als: past, present and future perspectives. Int J Antimicrob Fourth generation fluoroquinolones: new weapons in the
Agents 16:5–15, 2000 arsenal of ophthalmic antibiotics. Am J Ophthalmol 133:
2. Ball P: Quinolone generations: natural history or natural 463–6, 2002
selection? J Antimicrob Chemother 46:17–24, 2000 26. Ng EY, Trucksis M, Hooper DC: Quinolone resistance muta-
3. Ball P, Fernald A, Tillotson GS: Therapeutic advances of new tions in topoisomerase IV: relationship of the flqA locus and
fluoroquinolones. Exp Opin on Investigational Drugs 7:761– genetic evidence that topoisomerase IV is the primary target
83, 1998 and DNA gyrase the secondary target of fluoroquinolones
S78 Surv Ophthalmol 49 (Suppl 2) March 2004 BLONDEAU

in Staphylococcus aureus. Antimicrob Agents Chemother 40: 35. Wang JC, Lynch AS: Transcription and DNA supercoiling.
1881–8, 1996 Curr OpinGenet Dev 3:764–8, 1993
27. Norris S, Mandell GL: The Quinolones: History and Over- 36. Willmott CJ, Maxwell A: A single point mutation in the DNA
view. London: Academic Press Ltd; 1998: pp 1–22 gyrase A protein greatly reduces binding of fluoroquinolones
28. O’Brien TP, Maquire MG, Fink NE, et al: Efficacy of ofloxacin to the gyrase-DNA complex. Antimicrob Agents Chemother
versus cefazolin and tobramycin in the therapy of bacterial 37:126–7, 1993
keratitis. Report from the Bacterial Keratitis Research Group. 37. Yoshida H, Bogaki M, Nakamura M, et al: Quinolone
Arch Ophthalmol 113:1257–65, 1995 reistance-determining region in the DNA gyrase gyrB gene
29. Pan X, Fisher ML: Targeting of DNA gyrase in Streptococcus of Escherichia coli. Antimicrob Agents Chemother 35:1647–
pneumoniae by sparfloxacin: selective targeting of gyrase or 50, 1991
topoisomerase IV by quinolones. Antimicrob Agents Chemo- 38. Yoshida H, Bogaki M, Nakamura M, Nakamura S: Quinolone
ther 41:471–4, 1997 resistance-determining region in the DNA gyrase gyrA gene
30. Piddock LJ, Wise R: The effect of altered porin expression of Escherichia coli. Antimicrob Agents Chemother 34:1271–
in Escherichia coli upon susceptibility to 4-quinolones. J Antimi- 2, 1990
crob Chemother 18:547–9, 1986 39. Zechiedrich EL, Cozzarelli NR: Roles of topoisomerase IV
31. Schmidt FJ, Hofmann B, Hansen B, et al: Relationship be- and DNA gyrase in DNA unlinking during replication in
tween ciprofloxacin, ofloxacin, levofloxacin, sparfloxacin Escherichia coli. Genes Dev 9:2859–69, 1995
and moxifloxacin MICs and mutations in grlA, grlB, gyrA and
gyrB in 116 unrelated clinical isolates of Staphylococcus aureus.
J Antimicrob Chemother 41:481–4, 1998 The author reported no proprietary or commercial interest in
32. Smith A, Pennefather PM, Kaye SB, Hart CA: Fluoroquino- any product mentioned or concept discussed in this article. The
lones: place in ocular therapy. Drugs 61:747–61, 2001
33. Smith HJ, Nichol KA, Hoban DJ, Zhanel GG: Dual activity author has received a research grant and is on the speaker bureau
of fluoroquinolones against Streptococcus pneumoniae: the facts of numerous pharmaceutical companies, including Allergan, Inc.
behind the claims. J Antimicrob Chemother 49:893–5, 2002 Reprint address: Joseph M Blondeau, MSc, PhD, RSM (CCM),
34. Wang JC: DNA topoisomerases. Ann Rev Biochem 65:635– SM (AAM), FCCP, Royal University, 103 Hospital Drive, Saskatoon,
92, 1996 SK, S7N 0W8 Canada.