Bacteriostatic and Bactericidal Effect of Tigecycline on Leptospira spp.

Tigecycline is a relatively new antimicrobial, belonging to glycylcyclines with antimicrobial activity against a large spectrum of bacteria. Very few data are available on its effect on Leptospira spp., which consist in a bacteriostatic mechanism. The aim of this investigation was to evaluate the bacteriostatic and bactericidal effect of tigecycline on reference Leptospira strains belonging to 16 serovars. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined through the microdilutions method, and tetracycline was used as the control. Results showed that tigecycline had higher MIC and MBC values than tetracycline. Obtained MIC values were between 4 and 32 µg/mL, while MBC values between 16 and >128 µg/mL. Patoc (MIC: 4 µg/mL; MBC: 16 µg/mL) resulted in the most susceptible serovar, while the most resistant were Bataviae (MIC: 32 µg/mL; MBC: 64 µg/mL), Bratislava (MIC: 8 µg/mL; MBC 128 µg/mL), and Tarassovi (MIC: 8 µg/mL; MBC: >128 µg/mL). This is the first investigation focused on the effect of tigecycline against Leptospira spp. reference strains. Since tigecycline is used as a treatment for bacteremia and urinary tract disease, and these symptoms could be linked to Leptospira infection, the possibility of using this antibiotic as a treatment for leptospirosis should be evaluated. Further studies are needed to explore the possibility to use tigecycline for in vivo application against Leptospira.


Introduction
Leptospirosis is a zoonotic re-emerging disease caused by spirochetal Gram-negative bacteria belonging to genus Leptospira [1]. Recently, more than 260 antigenically distinct serovars of Leptospira spp. were isolated and identified, and the genus Leptospira was sub-grouped, based on it DNA relatedness to pathogenic, intermediate, and saprophytic species on the basis of the different levels of virulence exhibited for animals and humans. In detail, pathogenic Leptospira causes mild to severe infection, intermediate Leptospira may potentially be a pathogen and is responsible for mild infections, while saprophytic Leptospira spreads in environments and is a nonpathogen [2][3][4]. Recent studies highlighted the important role of intermediate and saprophytic Leptospira in the epidemiology of leptospirosis, because these can share habitats and could give rise to recombination events with pathogenic serovars [5][6][7].
Leptospirosis is diffused worldwide and occurs in tropical, subtropical, and temperate zones, representing public health problems, since its epidemiology involves humans and domestic and wild animals, which can be maintenance or accidental hosts [1,8,9]. Maintenance hosts, generally do not develop symptoms referable to disease and they represent important renal carriers, contributing to maintaining and sharing the infection, shedding Leptospira with urine in the environment. The accidental contact with Leptospira colonized urine represents the primary cause of incidental infection, producing clinical diseases [1,4]. For these reasons, Leptospira epidemiology is strictly connected to maintenance host species [10]. Therefore, specific Leptospira serovars are related to specific animal species, which involves specific maintenance hosts. Generally, for example, rodents are associated with Icterohaemorrhagiae and Ballum serogroups [11][12][13], swine with Pomona and Tarassovi serogroups [14][15][16][17][18], horse with Bratislava serogroups [19,20], and bovine and ovine with Sejroe serogroups [21,22].
Leptospirosis by pathogenic serovars can cause mild to severe infections in both humans and animals. Most cases are mild and they resolve spontaneously. [23]. Conventionally, the treatment for acute and severe leptospirosis consists of strong antimicrobial therapy with tetracycline, penicillin, or doxycycline [1,23]. Generally, molecules belonging to the tetracycline class are those mainly used in veterinary and human practices [1,[23][24][25].
Tigecycline is a relatively new antimicrobial, belonging to glycylcyclines, which derive from tetracyclines [26].
Tigecycline showed antimicrobial effects in vitro against many Gram-positive and Gram-negative bacteria, including multi-drug resistant strains (methicillin resistant Staphylococcus aureus, extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, multi-drug resistant Acinetobacter baumanii, and vancomycin-resistant Enterococcus spp.) [27,28]. The action of tigecycline consists of a bacteriostatic mechanism, which relies on the binding of 30 s ribosomal subunits of bacteria and blocking the entry of transfer of RNA [26].
Studies on the antimicrobial effects on Leptospira isolates or reference cultures are scarce, probably due to problems in the determination of antibiotic susceptibility [1,23]. A standardized microdilution method has been developed to accurately investigate the Leptospira antibiotic susceptibility, resolving the difficulties related to preventing the contamination of the medium, evaluating the bacterial growth, and reproducibility of the assay [29][30][31].
The aim of this investigation was to evaluate, through the microdilutions method, the bacteriostatic and bactericidal effects of tigecycline on reference Leptospira strains belonging to 16 serovars. Table 1 shows the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of tetracycline and tigecycline for different strains of Leptospira spp. Concerning tigecycline, obtained MIC values were between 4 and 32 µg/mL, while MBC values between 16 and >128 µg/mL. Patoc resulted in the most susceptible serovar, with MIC and MBC values of 4 and 16 µg/mL, respectively. Additionally, the most resistant serovars were Bataviae (32 µg/mL for MIC and 64 µg/mL for MBC), Bratislava (8 µg/mL and 128 µg/mL), and Tarassovi (8 µg/mL and >128 µg/mL).

Discussion
Treatments for Leptospira infections are based on the use of antibiotics belonging to a few specific molecules, generally tetracyclines. These antibiotics are conventionally used in medicine, thanks to no antimicrobial resistant events highlighted for Leptospira. The knowledge of antibiotic treatments against leptospirosis is strictly connected to in vivo clinical evaluation, usually in patients with severe and acute Leptospira infections [1,4,23]. Very few studies explored the efficacy and mechanisms of the action of new chemotherapeutic molecules [29][30][31]. This is the first investigation showing the in vitro effect of tigecycline against Leptospira spp.
The obtained results were consistent with other data available in literature, according to which tetracycline MIC values for Leptospira reference strains or isolates are, on average, between 0.25 and 6.25 µg/mL [29][30][31][32][33][34]. Considering that the results obtained for tetracycline were very similar to those reported in literature, it is correct to suppose that the results for MIC and MBC for tigecycline were also similar, not under-or overestimated. Since tetracycline and tigecycline belong to the same antibiotic class, it is reasonable to exclude any causes of strain attenuation due to the passages for culture maintenance, mutation, or acquisition of genes for antibiotic resistance.
Results showed that tigecycline had higher MIC and MBC values than tetracycline, used as the control. To the best of the authors' knowledge, no studies have focused on the tigecycline effect on Leptospira, excluding one in vivo assay. An administration of 5 mg/kg/day of tigecycline for 5 days was useful to reduce the presence of spirochetes in the blood of hamsters experimentally infected with 10 5 Leptospira interrogans serovar Canicola [35].
Although the tigecycline effect was investigated only in one in vivo study on Leptospira and in other studies on different spirochetes species, this investigation highlighted the bacteriostatic and bactericidal effect on Leptospira spp. strains of this antimicrobial molecule. Since this is the first investigation, further studies should be carried out to understand the antimicrobial effects against Leptospira, using reference strains and isolates, and to determine its potential employment for leptospirosis treatment or antimicrobial resistance. Moreover, further genomic studies are required to elucidate the molecular mechanisms responsible to elucidate the Leptospira antimicrobial susceptibility or resistance. Leptospira species don't have the ability to acquire genes for antimicrobial resistance during horizontal transfer and, if present, it is probably limited [24,31]. In humans, leptospirosis is a dead-end infection, where human-to-human transmission is very rare, considering that the infection is also usually monomicrobial, horizontal resistance gene acquisition from other bacterial species, as well as in the environment, very difficult [24]. Finally, the major causes of Leptospira antibiotic resistance seem to be related to spontaneous target gene mutations, as was in vitro evaluated for spontaneous mutation of the target gene 16S rRNA and rpsL for the development of spectinomycin and streptomycin resistance, respectively [40,41].

Leptospira spp. Strains
In this investigation, the following references of Leptospira strains were employed: Each strain was maintained in pure culture in the Ellinghausen-McCullough-Johnson-Harris (EMJH) medium, sub-cultured, and checked weekly for purity and viability.

Antibiotics
Tigecycline was purchased from Sigma-Aldrich (St. Louis, MO, USA), while tetracycline was purchased from Carlo Erba (Cornaredo, MI, Italy). Tetracycline was employed as the control to compare the results of tigecycline effects.

Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC)
The MIC determination was performed by the broth microdilution method, described by Liegeon et al. [31]. Leptospira cultures were quantified with spectrophotometry using optical density at 420 nm (Multiskan™ FC Microplate Photometer; Thermo Fisher Scientific, Haverhill, MA, USA) to reach a turbidity of 0.5 of the McFarland standard, which corresponds to approximately 1.5 × 10 8 cfu/mL [42,43]. In each well of 96-well plates, two-fold serial dilutions were performed in EMJH media, ranging from 128 to 0.25 µg/mL and from 64 to 0.125 µg/mL for tigecycline and tetracycline, respectively. The final volume in each well was 100 µL, including 5 µL of standardized strain. Two microplates were prepared for each strain: one to determine the MIC and the other for the MBC. They were incubated for 3 days at 30 • C, then 20 µL of resazurin sodium salt (Alfa Aesar, Thermo Fisher Scientific, Haverhill, MA, USA), diluted 1:30 in EMJH medium, was added to each well. The plates were incubated at 30 • C for another 2 days. A change in color from blue to pink indicated Leptospira growth. The MIC value was reported as the lowest concentration able to prevent a color change.
MBC was determined by plating 10 µL from each well (starting from the MIC value to higher antibiotic concentrations) in 1.5% EMJH agar. Plates were incubated at 30 • C for 4 days. The MBC value was reported as the lowest concentration able to prevent bacterial growth.
All tests were performed in triplicate and the results were expressed as the mode.

Conclusions
Tigecycline administration is usually employed in human patients for treatment of bacteria that causes pneumoniae and diabetic foot infections, but also for urinary tract infections and bacteremia [44,45]. Considering that bacteremia and urinary tract diseases could be symptoms compatible with Leptospira infection, the possibility of using this antibiotic as a treatment for leptospirosis should be evaluated. Data obtained in this in vitro investigation seem to be encouraging, and further studies are auspicial to explore the possibility of using tigecycline for in vivo application against Leptospira.
Funding: This research was funded by University of Pisa, grant "Fondi di Ateneo".

Conflicts of Interest:
The authors declare no conflict of interest.