Introduction

The fluoroquinolones, also known as quinolones, 4‐quinolones, pyridine‐beta‐carboxylic acids, and quinolone carboxylic acids, are a large and expanding group of synthetic antimicrobial agents. The first of these compounds, nalidixic acid, was initially described in 1962, introduced into clinical practice in 1963, and then approved for clinical use in 1965. Nalidixic acid had limited clinical application because of its poor absorption following oral administration, its moderate antibacterial activity (MICs of 4–16 μg/ml for Enterobacterales), high protein binding (92–97%), and poor patient tolerance.

Between the mid‐1960s and the early 1980s several other quinolones were approved for clinical use in humans, for example, oxolinic acid, pipemidic acid, piromidic acid, and flumaquine. These drugs exhibited increased antibacterial activity but still had limited absorption and distribution. In the 1980s, the addition of both a fluorine molecule at the 6 position of the basic quinolone structure and a piperazine substitution at the 7 position enhanced the antibacterial activity of these compounds, with activity against Pseudomonas aeruginosa and staphylococci. These modifications also increased the oral absorption and tissue distribution. The quinolone nucleus with the fluorine molecule gave the group the name “fluoroquinolones.” The first fluoroquinolone approved for use in human clinical medicine was norfloxacin, followed shortly thereafter by ciprofloxacin. The first fluoroquinolone approved for use in animals was enrofloxacin, which was approved for use in the United States in companion animals in 1988. The fluoroquinolones and their current clinical uses in veterinary medicine are listed in Table 17.1.

The World Health Organization (WHO) classifies the fluoroquinolones as Critically Important Antimicrobials for human medicine as they are needed for treatment for Campylobacter spp. infections, invasive disease due to Salmonella spp., and multidrug‐resistant Shigella spp. infections. The clear evidence that treatment of entire groups of animals with fluoroquinolones selects for resistance in important zoonotic pathogens mandates that these drugs should be used in veterinary medicine only when supporting laboratory data demonstrate that no suitable alternatives of less human health importance are available and use as mass medications should be restricted (Kenyon, 2021; McEwen and Collignon, 2018).

Chemistry

The fluoroquinolones, like sulfonamide and nitrofurans, are synthetic compounds so they are antimicrobials but not antibiotics. The first clinically approved 4‐quinolone‐type compound was nalidixic acid. Since the discovery of nalidixic acid’s antibacterial activities, more than 10 000 compounds have been designed from the parent bicyclic 4‐quinolone molecule. Clinically, nalidixic acid had several limitations, including a narrow spectrum of activity, poor pharmacokinetic properties, toxic effects, and a tendency to select for resistant organisms. Replacing the hydrogen atom at position 6 of the 4‐quinolone molecule with a fluorine atom resulted in increased activity against both Gram‐positive and Gram‐negative bacteria. The increased activity is attributed to increased penetration of the bacterial cell membrane. Substituting a piperazinyl ring for the methyl group at position 7 increased Gram‐negative activity, including antipseudomonal activity.

These modifications led to the development of the first broad‐spectrum fluoroquinolone, norfloxacin. Additional studies demonstrated that substantial changes in potency could be obtained by variations at the N‐1 and C‐7 positions. For example, ciprofloxacin is similar in structure to norfloxacin but has a cyclopropyl group in place of the ethyl group at N‐1. This substitution enhances ciprofloxacin’s Gram‐positive and Gram‐negative activity. This cyclopropyl group is also found on enrofloxacin, danofloxacin, pradofloxacin, and orbifloxacin (Figure 17.1). Difloxacin has a phenyl ring at position N‐1 that gives it enhanced activity against Gram‐positive bacteria, relative to enrofloxacin. Difloxacin also has a second fluorine atom in its structure, whereas orbifloxacin has a total of three fluorine atoms. These additional fluorine atoms do not appear to influence the antibacterial activity of these compounds.

Overall, there have been several chemical modifications at each of the eight positions in the 4‐quinolone molecule. Some increase absorption, some increase antibacterial activity, and others increase toxicity. For example, ciprofloxacin and enrofloxacin are similar molecules except for the ethyl group on the piperazinyl ring of enrofloxacin. This ethyl group enhances the oral absorption of enrofloxacin over ciprofloxacin but decreases its antipseudomonal activity.

Biological classification places the 4‐quinolones in four groups or generations (Pham et al., 2019). First‐generation quinolones are those with antibacterial activity restricted to the Enterobacterales (e.g., nalidixic acid and flumequine). Second‐generation quinolones have an extended spectrum of antibacterial activity. Most fluoroquinolones approved for use in people (including ciprofloxacin, norfloxacin, and ofloxacin) and all but one of the fluoroquinolones approved for use in veterinary medicine are second‐generation fluoroquinolones. Third‐generation fluoroquinolones have considerably improved activity against streptococci and obligate anaerobes. Examples of third‐generation fluoroquinolones approved for human use are sparfloxacin, grepafloxacin, and gatifloxacin. Pradofloxacin is the only third‐generation fluoroquinolone approved for use in animals. Fourth‐generation fluoroquinolones have all the activities of third‐generation drugs and extra anaerobic activity. Examples of fourth‐generation fluoroquinolones approved for human use are trovafloxacin and moxifloxacin. Newer compounds are being explored that optimize the various substitutions and allow for the fluorine atom at position 6 to be replaced, which may reduce adverse effects, decrease metabolism, and decrease interactions with other drugs. However, the emergence of resistance to the entire class of fluoroquinolones remains problematic.

Mechanism of Action

The bacterial chromosome is a continuous, circular, double‐stranded DNA molecule approximately 1000 times longer than the bacterium in which it is contained. In order for such a long molecule to fit into the cell, it is densely packed in a negative supercoil, twisted in the opposite direction to the right‐handed double helix of DNA. This supercoiled configuration is so highly strained that to improve function, the chromosome is divided into approximately 50 topologically independent domains.

Fluoroquinolones target two essential bacterial topoisomerase enzymes: DNA gyrase and DNA topoisomerase IV. Topoisomerase I is characterized by reactions involving single‐stranded DNA, whereas topoisomerase II is involved in reactions with double‐stranded DNA. Topoisomerase II, also known as DNA gyrase, consists of two subunits, GyrA and GyrB. The gyrA gene encodes two alpha‐subunits while the gyrB gene encodes two beta‐subunits; the active DNA gyrase is an A₂B₂ complex. DNA gyrase binds to DNA; a segment of approximately 130 nucleotide wraps around the DNA gyrase. This wrapped DNA is cleaved in both strands, forming a DNA–protein covalent bond between the GyrA subunit and the 5’‐phosphates of the DNA molecule. Another segment of DNA is passed through this double‐stranded break, which may then be resealed. The alpha‐subunit of the DNA gyrase is important in the breakage and reunion that allow for this relaxation of the DNA molecule. In multiple species of bacteria, it has been shown that the 4‐quinolone molecule interrupts the DNA breakage–reunion step by binding to the DNA gyrase–DNA complex at the interface between protein and DNA near the active site tyrosine and thus leads to defects in the negative supercoiling (Hooper and Jacoby, 2016).

Fluoroquinolones have a second intracellular target, DNA topoisomerase IV (Topo IV). This is a bacterial type II DNA topoisomerase and is also a multimeric protein composed of two ParC subunits and two ParE subunits, which exhibit sequence homology to GyrA and GyrB, respectively. This enzyme mediates relaxation of duplex DNA and the unlinking of daughter chromosomes following replication. However, unlike the DNA gyrase, Topo IV cannot supercoil DNA. Instead it is involved in the ATP‐dependent relaxation of DNA. It is a more potent decatenase than DNA gyrase.

Fluoroquinolones can differ in their potency for the two enzymes, with a general pattern among the drugs in clinical use that there is greater activity against DNA gyrase in Gram‐negative bacteria and greater activity against topoisomerase IV in Gram‐positive bacteria; but exceptions occur, and some quinolones have similar potency against both enzymes.

The effect of fluoroquinolones on bacterial proliferation suggests three mechanisms of cell killing (Martinez et al., 2006).

• Mechanism A is common to all fluoroquinolones. It requires RNA and protein synthesis and only acts on dividing bacteria. Mechanism A appears to involve the blocking of replication by the gyrase–quinolone complex on DNA.
  
• Mechanism B does not require RNA and protein synthesis and can act on bacteria that are not multiplying. Mechanism B correlates with dislocation of the gyrase subunits that constrain the ternary complex.
  
• Mechanism C requires RNA and protein synthesis but does not require cell division. Mechanism C may correlate with trapping of Topo IV complexes on DNA.

Antimicrobial Activity

The important features of the antimicrobial activity of the fluoroquinolones is their rapid bactericidal and concentration‐dependent killing. Targeting fluoroquinolone dosage to the MIC of the pathogen, as discussed below under Pharmacodynamic Properties, not only increases clinical efficacy but reduces the emergence of resistance.

The fluoroquinolones have excellent activity in vitro against a wide range of aerobic Gram‐negative bacteria, including the Enterobacterales, Actinobacillus pleuropneumoniae, Histophilus somni, Mannheimia haemolytica, and Pasteurella spp. including P. multocida. They are also active against Bordetella bronchiseptica, Brucella spp., Chlamydia/Chlamydophila spp., Mycoplasma spp., and Ureaplasma spp. Fluoroquinolones are active against rapidly growing mycobacteria isolated from dogs and cats. In general, pradofloxacin tends to be more active (i.e., lower MICs) against Gram‐negative bacteria than other veterinary fluoroquinolones (Silley et al., 2012). Activity against Pseudomonas aeruginosa is dependent on the fluoroquinolone, with ciprofloxacin being the most potent agent against this bacterium. For the most part, the first‐ and second‐generation fluoroquinolones are less active against Gram‐positive bacteria, especially enterococci, and have poor activity against anaerobic bacteria. Newer (third‐ and fourth‐generation) fluoroquinolones target this deficiency. For example, trovafloxacin, moxifloxacin, and gatifloxacin are newer human fluoroquinolones with good in vitro activity against obligate anaerobes. Pradofloxacin is active against anaerobic bacteria from dogs in cats including Clostridium spp., Bacteroides spp., Fusobacterium spp., and Prevotella spp.

In general, fluoroquinolones have poor efficacy against streptococci and monotherapy with enrofloxacin has been associated with streptococcal shock syndrome and necrotizing fasciitis in dogs (Ingrey et al., 2003). While most staphylococci initially show susceptibility to fluoroquinolones, methicillin‐resistant Staphylococcus spp. frequently show resistance to all fluoroquinolones (Kizerwetter‐Świda et al., 2016).

Fluoroquinolones exhibit a biphasic dose response curve (the Eagle effect) in that there is an optimum bactericidal concentration (OBC) above the pathogen’s MIC and beyond which the bactericidal activity decreases (Lewin et al., 1991). As the ratio of fluoroquinolone concentration to MIC increases from ≤1:1 to the OBC (usually shown to be approximately 10:1–12:1 but is drug‐bacterium dependent), bacterial killing increases and is usually very rapid.

As illustrated in Figure 17.2, when a strain of M. haemolytica is exposed to a fluoroquinolone at concentrations that are 25% of its MIC, the drug exhibits a slight stationary effect but then the bacterium resumes growth at a rate similar to that of the untreated control. As the concentration of the drug is increased above the MIC, there is a decrease in the number of viable organisms. For drug concentrations that are equivalent to the MIC, there is a slight decrease in the number of viable organisms but after 24 hours of exposure, the number of viable organisms has increased to more than what was in the starting suspension. This is without an increase in MICs. This suggests that this fluoroquinolone, at concentrations that are equal to the MIC, has a static effect on M. haemolytica. When the concentration of this fluoroquinolone is increased to four times the MIC, there is a nearly 4 log₁₀ reduction in the number of viable organisms within four hours of exposure. However, this killing effect stabilizes and then the organisms begin to proliferate, again without an increase in MIC. This is in contrast to the growth rate when the concentration of the fluoroquinolone is eight times the MIC. Under this circumstance there is a very rapid bactericidal effect, 7 log₁₀ reduction in viable organisms, and after a 24‐hour exposure there was no detectable regrowth of the bacterium. This suggests that at this concentration to MIC ratio, there was a 100% bactericidal effect.

The concentration‐dependent killing effect may plateau when the ratio of fluoroquinolone concentration to MIC reaches 15:1–20:1 and at ratios greater than 20:1 the fluoroquinolones may become bacteriostatic. The decrease in antibacterial activity at high drug concentrations is thought to be caused by the inhibition of RNA and protein. This implies that protein synthesis may be required for quinolone‐mediated cell death. Supporting this, protein synthesis inhibitors (such as chloramphenicol) and RNA synthesis inhibitors (such as rifampin) reduce fluoroquinolone effectiveness in bacterial killing in vitro.

Antimicrobial Resistance

Resistance to the fluoroquinolone occurs by target modification, decreased permeability, efflux, and/or target protection. Each of these fluoroquinolone resistance mechanisms can occur simultaneously within the same cell, thereby leading to very high resistance levels. To date, no mechanisms based on enzymatic inactivation/modification of fluoroquinolones have been discovered. Because fluoroquinolones are synthetic antimicrobials with no known natural analogues, it is less likely that this type of mechanism will emerge. Selection of resistant mutants with decreased permeability or efflux mechanisms generally means a 2–8‐fold increase in MIC, whereas alteration of the DNA gyrase binding site or target protection may result in high‐level resistance.

Resistance to one fluoroquinolone frequently results in resistance to all. This is especially true for the older drugs and for high‐level resistance. Fluoroquinolone resistance due to target mutations typically results in decreased susceptibility or resistance to other fluoroquinolones. Resistance due to alterations in permeability or activation of the efflux pump can confer resistance to other antimicrobial agents such as the cephalosporins, carbapenems, and tetracyclines even if these drugs have not been used in the patient (Hooper and Jacoby, 2016).

Because fluoroquinolones mediate DNA damage by binding to susceptible enzymes, fluoroquinolone resistance mutations are recessive. For topoisomerase‐mediated fluoroquinolone resistance to be transferred horizontally, an acquired mutated gene has to supplant the wild‐type gene. The development of fluoroquinolone resistance via mutations in topoisomerases has been studied extensively. Resistance is mediated primarily by target mutations in DNA gyrase (topoisomerase II), with secondary mutations in topoisomerase IV contributing to higher levels of resistance. Amino acid substitutions that result in bacterial resistance have been localized to a specific topoisomerase subdomain termed the quinolone resistance‐determining region (QRDR) within gyrA and parC. In E. coli, most mutations associated with quinolone resistance occur in the QRDR at serine 83 (Ser83) and aspartate 87 of gyrA, and at serine 79 and aspartate 83 of parC and at analogous sites in other species (Gebru et al., 2012). DNA sequence analysis of S. aureus and Streptococcus genes shows that the situation can be reversed in Gram‐positive bacteria, where topoisomerase IV (encoded by grlA and grlB) is the primary fluoroquinolone target. In both cases, mutations decrease the fluoroquinolone affinity for the enzyme/DNA complex and allow DNA replication to continue in the presence of fluoroquinolone concentrations that are inhibitory to wild‐type cell growth.

In Gram‐negative organisms, fluoroquinolone resistance typically develops in a stepwise manner. A single QRDR mutation, usually at Ser83, confers resistance to nalidixic acid and decreases susceptibility to fluoroquinolones (ciprofloxacin MICs may go from a wild‐type baseline of 0.015–0.03 μg/ml to 0.125–1 μg/ml). Secondary mutations in the gyrA QRDR lead to overt fluoroquinolone resistance (ciprofloxacin MICs ≥4 μg/ml). However, this does not hold true for all Gram‐negative bacteria. In Campylobacter spp., which lack topoisomerase IV, a single mutation in gyrA is sufficient to impart high‐level ciprofloxacin MICs (32 μg/ml) (Griggs et al., 2005). This feature helps explain the higher prevalence of resistance in Campylobacter, compared to E. coli, from food animals exposed to fluoroquinolones.

As indicated above, fluoroquinolone resistance may also be mediated by decreased permeability of the bacterial cell wall through altered outer membrane porins (OmpF) and by the activity of energy‐dependent efflux pumps. Most fluoroquinolones cross the Gram‐negative outer membrane through protein channels called porins, although some may diffuse directly across the lipid bilayer. Resistance due to decreased fluoroquinolone influx is generally reflected in low‐level changes in susceptibility and may explain differences in potency among different fluoroquinolone derivatives. Porin deficiency has been associated with quinolone resistance in E. coli and Pseudomonas. For example, mutations of the E. coli porin OmpF produce about a two‐fold increase in quinolone MICs.

However, it is difficult to experimentally assess the role of porins without also accounting for effects due to efflux. Permeability changes mediated by altered porins are often part of a coordinated cellular response to the presence of numerous toxic agents, which includes simultaneous upregulation of efflux. In E. coli, de‐repression in regulatory loci such as marA or soxS leads to decreased fluoroquinolone susceptibility via simultaneous upregulation of the AcrAB‐TolC efflux pump and downregulation of the OmpF porin. This mechanism confers decreased susceptibility to a large number of other antimicrobial agents in addition to fluoroquinolones. Analogous regulatory loci exist among other species of bacteria.

In antimicrobial efflux systems, membrane‐localized proteins actively pump drug from the cell before it can diffuse to its primary target within the active site of DNA gyrase. Because they are driven by the proton motive force, energy uncouplers can be used to study their role in resistance. The E. coli genome carries as many as 30 potential efflux pumps, many of which mediate antimicrobial efflux. Some are effective for specific agents, whereas others protect against a variety of structurally diverse compounds. In addition, a single bacterium may contain multiple efflux pumps (e.g., AcrAB and CmlA) that are capable of extruding the same antimicrobial agent. Constitutive and inducible efflux is a known mechanism of fluoroquinolone resistance in both Gram‐negative and Gram‐positive bacteria, and may be more important than secondary mutations in topoisomerase IV genes. For example, it has been shown that deletion of the gene encoding the inducible AcrAB efflux pump reduces ciprofloxacin MICs to near wild‐type levels in cells carrying topoisomerase mutations. In Campylobacter, where efflux mediated by CmeAB is constitutive, fluoroquinolone MICs in wild‐type cells are 3–4‐fold higher than those typical of E. coli. Insertional inactivation of CmeAB in C. jejuni reduces ciprofloxacin MICs to levels near that of wild‐type E. coli (0.003 μg/ml).

Bacterial fluoroquinolone resistance was once thought to disseminate exclusively via clonal expansion under selective pressure. A plasmid‐mediated quinolone resistance gene (qnrA) was first described in clinical isolates of Klebsiella pneumoniae

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