Efficacy of a New Recrystallized Enrofloxacin Hydrochloride-Dihydrate against Leptospirosis in a Hamster Model

ABSTRACT

A trial on Syrian hamsters (Mesocricetus auratus) infected with Leptospira interrogans serovar Canicola was established to compare treatment efficacies of daily intramuscular (i.m.) injections of either 10 mg/kg of 5% enrofloxacin (Baytril [BE]; Bayer Animal Health, Mexico) or the same dose of enrofloxacin hydrochloride-dihydrate (enro-C). Hamsters were experimentally infected via the oral submucosa with 400 microorganisms/animal, in a sequential time schedule aligned to the initial treatment day, and were treated in groups as follows: a group treated with 5% enrofloxacin daily for 7 days after 24 h of infection (group BE₂₄); a group treated as described for group BE₂₄ but with enro-C (enro-C₂₄); a group also treated with 5% enrofloxacin but starting at 72 h after infection (BE₇₄); a group treated as described for group BE₇₄ but with injection of enro-C (enro-C₇₄). An untreated-uninfected control group (group CG⁻) and an infected-untreated control group (group CG⁺) were assembled (n = 18 in all groups). Weights and temperatures of the hamsters were monitored daily for 28 days. After hamsters were euthanatized or following death, necropsy, histopathology, macroscopic agglutination tests (MAT), bacterial culture, and PCR were performed. The mortality rates were 38.8% in group BE₂₄ and 100% in group BE₇₄. No mortality was observed in group enro-C₂₄, and 11.1% mortality was recorded in group enro-C₇₄. The mortality rates in groups CG⁺ and CG⁻ were 100% and zero, respectively. Combined necropsy and histopathologic findings revealed signs of septicemia and organ damage in groups BE₂₄, BE₇₂, and CG⁺. Groups enro-C₂₄ and CG⁻ showed no lesions. Moderated lesions were registered in 3 hamsters in group enro-C₇₂. MAT results were positive in 83.3% of BE₂₄ hamsters (83.3%) and 100% of BE₇₂ and CG⁺ hamsters; MAT results were positive in 16.7% in group Enro-C₂₄ and 38.9% in group enro-C₇₂. Only 4/18 were PCR positive in group enro-C₇₂ and only 1 in group enro-C₂₄ (P < 0.05). It can be concluded that enro-C may be a viable option to treat leptospirosis in hamsters and that this may be the case in other species.

INTRODUCTION

Leptospirosis is considered the reemerging bacterial zoonosis most extensively distributed worldwide, and it affects most mammals (1, 2). More than a one million human cases are reported globally every year (3–5). Although it is a treatable disease, it is also potentially fatal in some cases, mostly due to delayed treatment (1). Mortality rates can reach more than 20% in humans and 48% in dogs (6). In Latin America, in sub-Saharan Africa, in the Middle East, and in some eastern countries, it is widely distributed in both rural and urban areas. There are over 260 pathogenic serovars; serological classification is based on differences in the carbohydrate component of the bacterial lipopolysaccharide (7). Different serovars are adapted to different wild or domestic animal reservoirs or hosts; hence, serovar recognition has epidemiologic importance. Infections are usually acquired through contact with leptospira-contaminated water reservoirs or by handling sick animals; access is facilitated through skin wounds or by contact with mucous membranes (8, 9). Once Leptospira reaches the bloodstream, rapid replication occurs in the absence of specific antibodies for 7 to 10 days (leptospiremia). Thereafter, it invades most body tissues and organs (10). Treatment during leptospiremia, usually by administering β-lactam antibacterial drugs, including benzylpenicillin G, ampicillin, amoxicillin, cefalexine, ceftriaxone, and cefalotine, is successful. Also, doxycycline and erythromycin have been reported to be reasonably effective (11). Treatment after leptospiremia has been established usually has to be more aggressive and prolonged and often has mediocre results (2).

Enrofloxacin, a fluoroquinolone derivative, has not been regarded as the first choice to treat leptospirosis, mainly because it shows relatively high (1 to 4 μg/ml) MIC values (12, 13) and because other antibacterial drugs reach therapeutic concentrations more easily (14). However, a new recrystallized form of enrofloxacin, enrofloxacin hydrochloride-dihydrate (enro-C), has already been described (15). This molecule shows higher water solubility than the parent compound, and it has already been shown to possess bioavailability superior to that seen with the reference enrofloxacin in broiler chicken (16) and hamsters (17). After its subcutaneous (s.c.) administration at a dose of 10 mg/kg of body weight in Syrian hamsters, enro-C showed remarkably high maximum concentration of drug in serum (C_max) and area under the concentration-time curve (AUC) values (17.3 ± 4.5 μg/ml and 459.2 ± 44.2 μg/ml/h, respectively [approximately 6-fold higher than the corresponding values for the reference preparation of enrofloxacin]). This is particularly interesting considering that infection of Syrian hamsters (Mesocricetus auratus) with Leptospira interrogans is one of the most widely accepted animal models for this disease (10, 18–20). Using the appropriate virulent Leptospira serovar, this species is highly prone to developing clinical leptospirosis, with complete lethality within 3 to 7 days in nontreated animals. Considering the data outlined above, the aim of this study was to assess the treatment efficacy of enro-C compared to that of the reference preparation of enrofloxacin, i.e., 5% Baytril, in the experimental infection of Syrian hamsters inoculated with a field isolate (strain LOCaS46) of Leptospira interrogans serovar Canicola.

RESULTS

Analysis of weight revealed a normal distribution on day zero in all groups (P < 0.05). Hamsters in the groups treated with 5% enrofloxacin daily for 7 days after 24 h of infection (group BE₂₄) and after 72 h of infection (BE₇₂) surviving the challenge exhibited statistically significant weight loss (P < 0.05). In contrast, hamsters in a group treated as described for group BE₂₄ but with enro-C (enro-C₂₄) did not show this trend and weights remained almost unchanged. In group enro-C₇₂, hamsters exhibited an initial decline in body weight, followed by a slight recovery, as can be appreciated in Fig. 1. As expected, all hamsters in the infected-untreated control group (group CG⁺) died and none of the hamsters in the untreated-uninfected control group (group CG⁻) died, and their weights remained almost unchanged. Body temperature was almost unchanged in group enro-C₂₄, and it started to fall sharply on days 2 to 4 in group enro-C₇₂, followed by full recovery. In groups BE₂₄ and BE₇₂, body temperature decreased as described for the CG⁺ group but never recovered, descending to low temperatures (30 to 30.8) thereafter until the end of the trial. These values have been depicted in Fig. 2. In that figure, no measurements can be observed in the BE₇₂ group for the last 2 days because all the animals in this group had died earlier. Figure 3 shows the cumulative values for mortality in all groups. Values for group CG⁻ are omitted because no fatalities were recorded such as occurred in group enro-C₂₄. In contrast, the CG⁺ group reached 100% mortality as shown in Fig. 3, which shows the high virulence of the strain used (LOCaS46).

FIG 1.

Mean ± 1 standard deviation (SD) weight values of Syrian hamsters after being inoculated with Leptospira interrogans serovar Canicola and treated as follows: for group BE₂₄, treatment with 5% enrofloxacin i.m. at a dose of 10 mg/kg/day for 7 days; for group enro-C₂₄, treatment as described for the BE₂₄ group but with enro-C; for group BE₇₂, treatment as described for group BE₂₄ but with the first injection of 5% enrofloxacin administered 72 h after inoculation; for group enro-C₇₂, treatment as described for group BE₇₂ but with enro-C. Values for the positive (infected-untreated) and negative (uninfected-untreated) control groups have been omitted for clarity. Initial n = 18 for group. P ≤ 0.01.

FIG 2.

Mean ± 1 SD temperature values of Syrian hamsters after being inoculated with Leptospira interrogans serovar Canicola and treated as follows: for group BE₂₄, treatment with 5% enrofloxacin i.m. at a dose of 10 mg/kg/day for 7 days; for group enro-C24, treatment as described for the BE₂₄ group but with enro-C; for group BE₇₂, treatment as described for group BE₂₄ but with the first injection of 5% enrofloxacin administered 72 h after inoculation; for group enro-C₇₂, treatment as described for group BE₇₂ but with enro-C. Groups CG⁺ and CG⁻ were positive (infected-untreated) and negative (not infected not treated) control groups, respectively. Initial n = 18 for group. P ≤ 0.01.

FIG 3.

Cumulative mortality of Syrian hamsters after inoculation with Leptospira interrogans serovar Canicola and treatment as follows: for group BE₂₄, treatment with 5% enrofloxacin i.m. at a dose of 10 mg/kg/day for 7 days; for group enro-C₂₄, treatment as described for the BE₂₄ group but with enro-C; for group BE₇₂, treatment as described for group BE₂₄ but with the first injection of 5% enrofloxacin administered 72 h after inoculation; for group enro-C₇₂, treatment as described for group BE₇₂ but with enro-C. Group CG⁺ consisted of untreated infected hamsters. No mortality was observed in the uninfected-untreated CG⁻ group. For clarity, data are not shown. Initial n = 18 for group. P ≤ 0.01.

Clinical signs of leptospirosis became evident on day 3 to day 5, with a marked decrease of physical activity or prostration, shaggy hair, hyporexia or anorexia, polyuria, polydipsia, hematochezia, hematuria, epistaxis, nystagmus in some animals, and seizures. Note that all hamsters in groups CG⁻ and enro-C₂₄ survived and that 88.9% (16/18) of those in group enro-C₇₂ did so. Mortality reached 55.6% (10/18) in group BE₂₄ and 100% in groups BE₇₂ and CG⁺. Gross anatomical findings at necropsy in groups BE₂₄, BE₇₂, and CG⁺ exhibited evidence of septicemia and organ damage. Lesions were more discrete in the hamsters that survived until day 28. In contrast, all 18 hamsters in group enro-C₂₄ showed no lesions on day 28, and moderate lesions could be observed in only 3 hamsters in the enro-C₇₂ group on day 25 (Fig. 3).

Necropsy data and histopathologic changes in groups BE₂₄, BE₇₂, and CG⁺ included moderate to severe diffuse pleural hemorrhage, collapsed alveoli, decreased airspace, and moderate levels of alveolar inflammatory cell infiltrate. In liver, hepatic necrosis, vacuolar degeneration of hepatocytes, and plasma cell infiltration were the dominant traits found (Fig. 4). Also, binucleate hepatocytes were observed in most animals. Interstitial nephritis, tubular necrosis, severe vacuolar degeneration, and glomerular hemorrhage were recorded in these animals, whose general aspect was characterized by jaundice, as shown in Fig. 5. Also, moderate to severe edema and hemorrhage were observed in the gastrointestinal (GI) tract. In contrast, none of these changes were observed in hamsters from groups enro-C₂₄ and CG⁻ and the mortality level among the hamsters examined from group enro-C₇₂ was greatly diminished (2 hamsters). A binary logistic regression model (confidence interval [CI] = 0.877 to 8.444) showed that hamsters in group BE₂₄ had a 2.718 odds ratio of organ and tissue lesions compared to the hamsters in group enro-C₂₄ (P < 0.05). This value increased to 5.6389 (CI = 2.543 to 12.659) when comparing the BE₇₂ group to the enro-C₇₂ group (P < 0.05).

FIG 4.

Macroscopic lesions found in a hamster of the group treated with the reference preparation of enrofloxacin at 72 h after infection (group BE₇₂). Jaundice is evident; the bladder is full and contains blood. There is renal congestion and moderate-to-severe lung hemorrhaging.

FIG 5.

Comparison of histological sections of renal tissue of hamster in group ER72 (A and C) and group enr-C₇₂ (B and D). Hemorrhages and tubular and glomerular destruction can be observed in panels A and C.

On day 28, it was possible to observe, by means of dark-field microscopy, Leptospira microorganisms in samples of kidneys from 12 animals in group BE₂₄, 16 in group CG⁺, and 15 in group BE₇₂. By comparison, using the same technique, Leptospira microorganisms could be seen in only 3 hamsters in group enro-C₇₂ and in none in groups enro-C₂₄ and CG⁻. Congruently, after bacteriological culture of samples recovered after mortality in hamsters from groups BE₂₄, BE₇₂, and CG⁺, Leptospira microorganisms could be recovered in 50%, 77.8%, and 100%, respectively, including the hamsters that died before reaching day 28 plus those sacrificed at the end of the trial. No isolation of Leptospira microorganisms was possible in animals from groups enro-C₂₄, enro-C₇₂, and CG⁻.

Macroscopic agglutination results were positive in 83.3% (15/18) of the BE₂₄ hamsters and in 100% of the hamsters from the BE₇₂ and CG⁺ groups. The same test gave positive results in 16.7% (3/18) and 38.9% (7/18) of the enro-C₂₄ and enro-C₇₂ hamsters, respectively (Fig. 6).

FIG 6.

Agglutination test titers of Syrian hamsters after inoculation with Leptospira interrogans serovar Canicola and treatment as follows: for group BE₂₄, treatment with 5% enrofloxacin i.m. at a dose of 10 mg/kg/day for 7 days; for group enro-C24, treatment as described for the BE₂₄ group but with enro-C; for group BE₇₂, treatment as described for group BE₂₄ but with the first injection of 5% enrofloxacin administered 72 h after inoculation; for group enro-C₇₂, treatment as described for group BE₇₂ but with enro-C. Tests were carried out on day 28 on survivors or postmortem in animals that died before that date. n = 18. P ≤ 0.0.1.

Endpoint PCR test results for the group CG⁺ hamsters and for the hamsters from both BE groups were positive in all instances, and only 4 of 18 analyses gave positive results in group enro-C₇₂. The results were positive for only one hamster in group enro-C₂₄ (see Fig. 7). The binary logistic regression model revealed that renal samples from the BE groups showed a 5.885 higher likelihood of finding DNA of leptospira than the samples from the enro-C groups (P < 0.05; CI, 2.379 to 11.785).

FIG 7.

Results of the endpoint PCR evaluation of the 18 animals on day 28 or at the time of death in groups ER₇₂ (A) and enr-C₇₂ (B) and in control groups (C). Initial n = 18 for group. P ≤ 0.01.

DISCUSSION

The enro-C treatment scheme proposed in this study showed a 100% clinical cure rate and an approximately 94% bacteriological cure rate in the cases in which the drug is administered every 24 h after infection and thereafter daily for 7 days. Moreover, animals showed minimal discomfort when enro-C was administered and no apparent lesions were observed. In contrast, severe tissue damage was observed in animals treated with 5% enrofloxacin. A 26% weight loss in hamsters treated with 5% enrofloxacin was observed. In contrast, the administration of enro-C prevented the appearance of this sign of leptospirosis. Also, the body temperature remained virtually unchanged in enro-C-treated hamsters, while 5% enrofloxacin-treated hamsters showed a fall of 2.2°C in body temperature. Again, minimal to absent macroscopic and histopathologic lesions were observed in hamsters treated with enro-C. Administration of enro-C 72 h after infection resulted in deaths in 11%, a fact that emphasizes the susceptibility of hamsters to leptospirosis and the need for rapid antimicrobial intervention against leptospirosis. In hamsters, Leptospira microorganisms can rapidly reach target organs after inoculation. This accounted for the reduced cure rate when treatments were administered after 72 h following inoculation (21).

To the best of our knowledge, there have been no reports of studies of leptospirosis in hamsters showing successful treatment with fluoroquinolones. For example, ciprofloxacin therapy resulted in survival rates of 90% when given at 50 mg/kg of body weight/day and 60% when given at 25 mg/kg/day. All animals treated with ciprofloxacin at 5 mg/kg/day died prior to the end of the study. Nevertheless, daily doses of ciprofloxacin at 25 and 50 mg/kg were associated with severe and, often, lethal adverse drug reactions. Using gatifloxacin at a dose rate of 50 mg/kg/day, a survival rate of 60% can be obtained. However, severe gatifloxacin-associated diarrhea was always observed (22). In fact, lately, only macrolides have been used in challenge model studies for the treatment of leptospirosis in the hamster model. In spite of some methodological differences, most of those studies followed similar guidelines, and their results can be compared with the results obtained in this trial. In one study, a 100% survival rate was achieved with clarithromycin at a dose of 60 mg/kg. However, the authors failed to disclose whether the success rate was due to clinical or bacteriological cure and severe adverse effects also occurred with the administration of that high dose. With the administration of a conventional dose of 20 mg/kg, a survival rate of only 30% was achieved (23). With the daily administration of 15 mg/kg of telithromycin, a new macrolide derivative, a 90% survival rate was achieved. However, at that dose, seizures were reported in some animals (23). With the oral administration of doxycycline, 80% to 90% clinical cure rates are observed. However, this antibacterial drug is known to induce a great variety of adverse reactions, mainly, gastrointestinal irritation, vomiting, diarrhea, and depression (23).

Enrofloxacin is not customarily considered an option for the treatment of leptospirosis in domestic or laboratory species (14, 24). Levels of in vitro activity do not appear to differ between enrofloxacin and enro-C treatments (15–17). Hence, it would appear that enrofloxacin shows insufficient tissue distribution compared to enro-C, thus failing to reach the necessary bactericidal concentrations against Leptospira spp. This correlates well with the reportedly much higher bioavailability of enro-C (17) and stands out as the most likely explanation for the observed differences between enrofloxacin and enro-C in their levels of clinical and bacteriological efficacy. Pharmacokinetic/pharmacodynamic (PK/PD) ratios are noticeably higher for enro-C, as presented by Carrascosa et al. (17). That is, intramuscular (i.m.) administration of 10 mg/kg of enro-C resulted in a noticeably high C_max value (17.3 ± 4.5 μg/ml) and also in a large value for the AUC from h 0 to h 24 (AUC_0–24) (459.2 ± 44.7 μg/ml/h). The MIC₉₀ values for enro-C and standard enrofloxacin were found to range from 0.25 to 0.5 μg/ml in this trial, comparable to previously established data for this laboratory (17). Considering this, the desired PK/PD ratios for enro-C (C_max/MIC and AUC_0–24/MIC) would have exceeded the stipulated ones in the formal literature for Leptospira spp., i.e., ≥10 and ≥125, respectively (12, 25, 26). Thus, the contrasting clinical and bacteriological cure rates of enro-C and the reference enrofloxacin found in this study may be based on better PK/PD ratios for enro-C for treatment of leptospirosis in hamsters, as proposed by Carrascosa et al. (17). Notwithstanding the data presented above, note that the exceptional efficacy of enro-C found in this study applies only to hamsters and to the use of the Leptospira challenge model described here. However, it is tempting to evaluate the efficacy of enro-C in clinical outbreaks of leptospirosis in other species. The clinical scenario is bound to be different, particularly because pharmacological interventions during the first stages of leptospiremia in veterinary medicine are rare, due to the inherent difficulties of an early diagnosis. Studies on the clinical efficacy of enro-C in canine and bovine species and in birds and other exotic pets such as ferrets appear to be a logical next step.

MATERIALS AND METHODS

All study procedures and animal care activities were conducted in accordance with a Mexican official regulation (NOM-062-ZOO-2001) (27) under the oversight of the Institutional Committee of Research, Care and Use of Experimental Animals of the Faculty of Veterinary Medicine of the National Autonomous University of Mexico.

A total of 108 8-week-old Syrian hamsters were kept in isolation conditions in individual cages at 22°C and 40% humidity, with a 12-h/12-h dark/light cycle and with feed and water ad libitum. In order to mimic the natural route of infection, all animals, except those from group CG⁻, were inoculated with Leptospira interrogans serovar Canicola strain LOCaS46 (400 bacteria/10 μl) through the submucosal route in the cheek pouches under conditions of isofluorane sedation. For this strain, the 50% lethal dose (LD₅₀) ranges from 10 to 10,000 bacteria/μl (28). The inoculation was done 24 h or 72 h after the initial treatment as follows: group BE₂₄ (n = 18) was treated with 5% enrofloxacin administered i.m. at a dose of 10 mg/kg of body weight/day for 7 days; group enro-C₂₄ (n = 18) was treated as described for the BE₂₄ group but with enro-C; group BE₇₂ (n = 18) was treated as described for group BE₂₄ but with the first injection of 5% enrofloxacin administered 72 h after infection; group enro-C₇₂ was treated as described for the BE₇₂ group but with enro-C; a positive-control group (CG⁺) was established with infected-untreated animals (n = 18) receiving an injection of saline solution 24 h later; finally, a negative-control group (CG⁻) consisting of uninfected-untreated hamsters (n = 18) also received an injection of saline solution 24 h later.

The enro-C batch was synthesized as described in Mexican patent number MX/a/2013/014605 (Instituto Mexicano de la Protección Industrial, submitted in December 2013, Mexico City), based on good manufacturing practices (GMPs) and according to a Mexican regulation (NOM-012-ZOO-1993) (29), and a fine white powder with a clearly defined structure was thus obtained as shown in Fig. 8 (15, 16). The powder was diluted with injectable water to achieve a 5% fine suspension just before injection.

FIG 8.

(A) Molecular structure of enro-C with displacement ellipsoids at 50%. Hydrogen atoms have been omitted for clarity. (B) Chemical structure of base enrofloxacin.

Evaluation of Leptospira virulence was carried out as suggested by Adler and de la Peña Moctezuma (30) as follows. Individual hamster weight and body temperature were recorded every 24 h for 28 days (Fluke-568EX infrared digital thermometer; Grainger, Mexico) (160 by 100 by 4 mm). On day 28, surviving animals were euthanized with an overdose of isofluorane in a closed chamber. Necropsies were performed on these hamsters and in those that died during the 28 days of the test. Animals about to die were left undisturbed. Necropsy and histopathology analyses of all animals, whether deceased before day 28 or sacrificed on completion of the trial, were carried out according to the methodology previously detailed by Presnell and Schreibman (31). Tissue samples from kidneys, livers, and lungs were obtained and fixed in 10% buffered formalin solution. Then, samples were routinely processed and embedded in paraffin. Sections of 4 μm thickness were cut and stained with hematoxylin-eosin using a standard technique and studied under a light microscope (31). Histopathological findings were characterized and categorized by a pathologist. Additionally, samples from these organs were homogenized and a drop of EMJH culture medium (Difco, Proveedor Cientifico S.A., Mexico) was added. These samples were utilized to directly search for Leptospira microorganisms under a dark-field microscope (19). Also, two droplets of each tissue homogenate were placed in Fletcher's and Stuart's EMJH modified media (Difco) and incubated at 30°C for 90 days, with checking for the possible presence of Leptospira microorganisms performed every week.

Macroscopic agglutination tests (MAT) were performed to detect positive serum samples on day 28, as detailed by Faine et al. (32). In addition, endpoint PCR analyses were performed on all subjects as follows: extraction of DNA from kidney samples was based on the use of a QIAamp DNA minikit (Qiagen), and the concentration and purity of DNA were evaluated using a Nanodrop 2000 kit (Thermo Fisher Scientific, Waltham, MA, USA). Then, primers MILL2509f and MILL2510r were used to amplify DNA from Leptospira interrogans serovar Canicola from strain 203PB, gen LipL32 (major outer membrane lipoprotein). Primers were kindly provided by the Institute of Biotechnology at the National Autonomous University of Mexico (UNAM).

Statistical analyzes were carried out by means of the generation of Kaplan-Meier curves through the use of the log rank test. Body weight and temperature were analyzed using a generalized linear model for longitudinal data. A binary logistic regression model was used for analysis of risk in organ and tissue lesions, and PCR test P values of ≤0.01 were considered significant.