Enrofloxacin is a zwitterionic molecule with a pKa₁ = [5.88 - 6.06] and a pKa₂ = [7.70 - 7.74] [5] . The lowest

pKa is due to the carboxyl acid group and the second to the basic tertiary amine. So enrofloxacin doesn’t bear charge between this two pH. Moreover it is a lipophilic molecule with a logP of 4.70 at a pH of 7 [6] , but 1.88 ± 1.43 with ACDLabs.

The Active metabolite of enrofloxacin, ciprofloxacin (Figure 1(b)) or 1-Cyclopropyl-6-fluoro-1, 4-dihydro-7- (1-piperazinyl)-4-oxo-3-quinoline carboxylic acid, which is available as a drug product on veterinary and human market, is a multiple acid with pKa₁ = 5.15 and pKa₂ = 8.25 [7] . Ciprofloxacin is less lipophilic than enrofloxacin with a logP of −1.11 at a pH of 7.4 [8] .

Enrofloxacin, like the other quinolones, has two mains targets of the topoisomerase family. Although these proteins exist in eukaryotes cells, quinolones have less affinity for eukaryotes’ topoisomerases than for the DNA Topoisomerase II (Gyrase) and the DNA Topoisomerase IV (Topo IV) two major bacterial topoisomerase [9] . The Gyrase and the Topo IV are two tetramers (Figure 2(a)) formed respectively of two GyrA and two GyrB and of two ParC and two ParE. Moreover GyrA and GyrB are homologous respectively with ParC and ParE [10] .

The Gyrase has an important role in bacteria’s life by modifying the topology of the spiral DNA. Indeed, the positive supercoiling stabilizes the DNA and the strands’ separation becomes more difficult [11] . Moreover transcription generates a positive supercoiling accumulation that can stop transcription. This positive supercoiling can be released by the Gyrase, which enhances transcription [12] . To do this, the Gyrase binds and wraps around itself a strand of DNA (Figure 2(b)) with the help of the C-terminal domain [13] and cleaves it (Figure 2(c)) with mediating of the catalytic tyrosine Tyr 122 [14] of each GyrA. Tyrosines form covalent phosphotyrosyls with each 5’ phosphoryl terminus of both strands, which remain binding during the reaction [15] . This reaction forms a gap in sequence (called G-DNA). The GyrB part catches the other DNA sequence (called transported DNA or T-DNA). The T-DNA is passed through the opened G-DNA (Figure 2(d)) [16] . The G-DNA is closed in an ATP-dependent reaction (Figure 2(e)) [17] . Of this reaction results an adding of negative coiling.

The Gyrase is able to do an intermolecular strand passage at the end of the replication like the Topo IV but

without wrapping the DNA as the Topo IV, this is the major difference between reactions of both enzymes [18] . Topo IV catalyzes the segregation of the two daughter DNA molecules after the replication [19] more efficiently than Gyrase [20] .

The penetration in the bacteria differs between Gram-positives and Gram-negatives bacteria. For Gram-negative bacteria, fluoroquinolones pass the outer membrane mainly through porins [21] [22] . The trimeric OmpF porin is mainly used by fluoroquinolone, this pathway can be modulated by Mg²⁺ [23] . But it seems that some quinolones can promote a pathway by interacting with the Lipopolysaccharide (LPS) and create a lipophilic passage [24] . For Gram-positive bacteria the diffusion process is the main uptake pathway [25] .

In the case of mycobacteria the high lipid level of the membrane allows fluoroquinolones to diffuse through membrane [26] .

After the entrance in the bacteria, quinolones have two effects on the bacteria: bacteriostatic at low concentration level or bactericide at high concentration level. Rapidly after the Gyrase has formed a complex with the DNA strand, two quinolones bind with this complex, before the DNA’s cleavage [27] . This binding is reversible and induces a conformation modification in Gyrase [28] . This modification induces a cleavage of DNA in a particular location and forms a cleaved complex [29] [30] . At this step the action is still reversible, but reduces rapidly the activity of replication and has a bacteriostatic effect [31] .

The next two pathways are possible one for bacteriostatic concentration and the other for bactericide concentration. For bacteriostatic concentrations, quinolones induce the SOS regulon controlled by the repressor lexA [32] [33] . Activation of the regulon induces the sfiA gene which codes for an inhibitor of cell division and bacteria form a long filamentous structure. This effect is still reversible [34] . This filamentous structure contributes to the death of bacteria [35] .

For bactericide concentration quinolones induce a chromosome fragmentation by creation of a suicide factor or by destabilization of a GyrA dimer [35] . The fragmentation can induce a rapid death or, by reassociation of the GyrA dimer, genomic mutation in bacteria [36] .

The resistance to fluoroquinolones in general, is today a main subject for the use of antibiotic substances in veterinary medicine and was the subject of an EMA report [4] . Many studies show an augmentation in the development of resistances to enrofloxacin [37] [38] . Moreover, another study shows the increase of resistance of Escherichia coli to quinolones and fluoroquinolones after using of enrofloxacin in calves [39] .

The mechanism of resistance to fluoroquinolones is based on many pathways: the change of targets, the protection of target, the lower expression of target, the improvement of the efflux pumps, and inactivation of fluoroquinolones. These mechanisms can be chromosomal or plasmide-mediated (Table 1) [40] .

The most common mechanism is the mutation of Gyrase. In the subpart GyrA the mutation is mainly between Ala67 and Gln 106 for E.coli and other bacteria [41] [42] . The mutation is near the GyrA active site which is called Quinolone Resistance-Determining Region (QRDR). For GyrB two mutation are reported at Asp426 and Lys447 [43] . Only one Gyrase mutation is able to increase the Minimum Inhibitory Concentration (MIC) up to 64 times [41] , but the GyrB mutation seems to be less effective in resistance against nalidixic acid than the GyrA mutation [44] .

The mutation of Topo IV is also described but, until today, always with at least one Gyrase mutation. Therefore, these mutations seem to be developed in a second time to increase the resistance more than only Gyrase’s mutations. A QRDR can be also characterized in the parC of topoisomerase IV between Tyr 57 and Glu84 [45] .

The protection of target can be attributed to a qnr gene often present on a Plasmide-Mediated Quinolone Resistance (PMQR) or in the chromosomal DNA. The Qnr protein is a dimere which binds with Gyrase and decreases the binding of quinolones with DNA-Gyrase-Qnr complex. But it seems that the Qnr protein reduces the interaction between DNA and Gyrase [46] . Another theory is that the Qnr protein destabilizes the DNA-Gyrase- quinolone complex and promotes the DNA repair after cleavage [47] .

An hypothesis of resistance is the slow growth of cell, bacteria growing slowly seem to resist better, but this mechanism is not known today [40] [48] .

Although resistances are often attributed to the Gyrase or Topo IV mutations, decrease of influx and increase of efflux mechanisms are important pathways of resistance common to a large number of unrelated antibiotics [49] . This defines a Multiple Antimicrobial Resistance (MAR) phenotype. E. coli bearing a MAR operon has higher resistance for the quick death by fluoroquinolones [50] . This operon can induce a decrease of OmpF pumps [51] and an increase of efflux with AcrAB protein [52] . The decrease of porins can also be attributed to PMQR [53] .

Given the rapidity of development of resistance, we have to consider with the MIC, the Mutant Prevention Concentration (MPC). MPC is a recent indicator which is not well standardized. Commonly MPC50 is the concentration where 50% of the colonies doesn’t contain resistant mutant after 72 hours of incubation. To count the mutants, a Polymerase Chain Reaction (PCR) is used but only known resistant mutation is tested [54] . Between MIC and MPC there is a window where enrofloxacin is effective but also where mutants are selected.

The data recorded in Table 2 show that the window between the MIC and the MPC can be very important with a MPC/MIC between 1.6 and 64. This high difference can explain the explosion of resistance against enrofloxacin and might be a base to define new guidelines and dosage for enrofloxacin.

There is high variation of bioavailability after oral administration of enrofloxacin between polygastric and monogastric animals from 10% to 80% [55] [56] . This has a direct consequences on the galenic, oral presentations are reserved for pigs, poultry, calves, and carnivores and only injectable solutions are available for cattle. Moreover, bioavailability depends on whether the animals are fed or fasted [55] [57] , and of the presence of ion [58] . These interferences can be explained by the formation of a complex between cation and fluoroquinolone which cannot permeate through the digestive barrier [59] . Another consequence of this complex is the influence of the hardness of water notably in poultry farming where the dilution in water is used [60] . But lipophilic compounds in food can enhance oral bioavailability of fluoroquinolone [61] .

In addition to passive diffusion allowed by enrofloxacin lipophilicity, active transporters have also an important role in intestinal absorption [62] . But these transporters are also important to eliminate fluoroquinolones [62] .

The intramuscular bioavailability is 96% with a maximum concentration 3 hours after administration [3] . Intramuscular bioavailability can be enhanced by solid lipophilic nanoparticles, this technic enhances the duration of enrofloxacin in plasma [3] .

After administration a high part of enrofloxacin is metabolized into ciprofloxacin in most of species (Table 3). Enrofloxacin has an active metabolite, ciprofloxacin [63] , obtained by deethylation of the ethyl on the piperazin ring. Other metabolites are obtained but they don’t have antimicrobial effects [64] .

Only poultry does not have a huge part of enrofloxacin metabolized in ciprofloxacin. The first hepatic pass doesn’t have a lot of effect, only 7% of enrofloxacin is metabolized [65] . Moreover ciprofloxacin seems a more potent drug than enrofloxacin [66] [67] . These elements are in favor to grant a more important place to ciprofloxacin in the effects of enrofloxacin-based drugs.

Distribution of enrofloxacin and ciprofloxacin to tissues depends on drug’s free concentration, which depends itself on the concentration of protein and the strength of this biding.

The difference between ciprofloxacin and enrofloxacin protein binding (Table 4) are in favor of the theory of enrofloxacin’s role of prodrug. Indeed, in some species ciprofloxacin is less bound with proteins, so it is able to be more available to be effective. Moreover, enrofloxacin can interact with other protein-binding drugs for instance it increases the clearance of flunixin meglubine [68] .

As described in Table 5, the volumes of distribution of enrofloxacin and ciprofloxacin in different species are all higher than 1. So enrofloxacin and its metabolite diffuse strongly in tissues, and molecules can be present in cells, so being inactive. Contrary to what we might expect considering its protein binding, ciprofloxacin doesn’t diffuse in tissues more than enrofloxacin. Data of Table 6 suggest a high affinity of drugs for lung and kidney, even if data confirm enrofloxacin’s and ciprofloxacin’s good diffusion.

^*elimination half-live, ^**clearance, ^***volume of distribution at the steady state. Enro: enrofloxacine, Cipro: ciprofloxacine.

^†Unit of C is µg/mL and unit of AUC is µg.h/mL; ^‡Unit of C is µg/g and unit of AUC is µg.h/g.

Elimination parameters show great difference between species (Table 5), especially for pigs where the elimination half-life is high with 26 h [69] . Moreover, for chickens and pigs, clearance of ciprofloxacin is five times higher than enrofloxacin’s clearance and their enrofloxacin clearances are lower than other species. This differences might be attributed to a difference of elimination way.

Enrofloxacin’s elimination way is mainly renal. This has been proved in rat by nephrecotomizing and comparing with CCl₄ hepatic impairment [70] and in goat with probenecid [71] . Probenecid has been shown to reduce the renal clearance of fluoroquinolones [72] . On the other hand, ciprofloxacin’s elimination is both hepatic and renal [73] . For both molecules there is an intestinal recirculation via the bile excretion, moreover, two hour after given, there is no significant difference in the concentration of enrofloxacin in the intestinal content between oral and intramuscular administration of enrofloxacin [74] . Lactation can influence significantly enrofloxacin’s and ciprofloxacin’s elimination by increasing two folds clearance in dairy cows comparative to steers [75] . It might be explained by an ionic trap.

The first veterinary product based on enrofloxacin was launched by the laboratory Bayer in 1991 (marketing authorisation in 1991) under the trade name Baytril^® and it was an oral form for poultry [3] . Now, many veterinary products on the basis of enrofloxacin are available on the market with at least thirty four veterinary medicines under different forms including oral and injectable forms, tablets or bolus [3] . There are many target species including domestic carnivores (dogs and cats), farm animals (cattle, pigs, poultry) and even exotic pets since 2010 [3] .

Enrofloxacin is indicated in the treatment of local and systemic diseases caused by a wide range of Gram-negative and Gram-positive bacteria [4] [76] . The most important indication of enrofloxacin in all of the species is the treatment of respiratory infections but it is also indicated in the treatment of digestive, urinary, joint, genital, mammary and dermal infections [3] [77] .

However, enrofloxacin is a third generation fluoroquinolone with a very large spectrum of activity so it has to be reserved to second intention. Indeed, in order to avoid fluoroquinolone resistances, it is important to reserve the use of enrofloxacin to infections resistant to over antibacterial agents and if possible under a susceptibility study [4] .

In the field conditions, enrofloxacin is often off-label used empirically to prevent uterine infections in susceptible embryo-transfer mares. In fact, a conventional dose (5 mg/kg body weight) given pre-breeding followed by two further doses at 36 - 48 h post breeding are supposed to prevent bacterial adherence and provide effective bactericidal concentrations in utero [78] .

Enrofloxacin is often used in aquaculture in Indonesia, Thailand and Vietnam. Indeed, because of the non-hygienic and stressful conditions in aquaculture facilities, the risk of bacterial infections is high and motivates the widely use of antibiotics in fish feed for prophylactic and therapeutic purpose [79] .