Leptospirosis is a zoonotic re-emerging disease caused by spirochetal Gram-negative bacteria belonging to genus Leptospira
]. 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
]. 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
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
]. 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
]. 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
], swine with Pomona and Tarassovi serogroups [14
], horse with Bratislava serogroups [19
], and bovine and ovine with Sejroe serogroups [21
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
]. Generally, molecules belonging to the tetracycline class are those mainly used in veterinary and human practices [1
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
]. 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
]. 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
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.
shows the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of tetracycline and tigecycline for different strains of Leptospira
For tetracycline, MIC values were between 0.5 and 2.0 µg/mL, while MBC values were between 16 and 64 µg/mL. Canicola, Autumnalis, and Hebdomadis resulted in the most susceptible serovars, with MIC values of 0.5 µg/mL, while MBC values were between 16 and 32 µg/mL. On the other hand, Tarassovi and Ballum resulted in the most resistant serovars with MIC and MBC values of 2 and 64 µg/mL, respectively.
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).
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
]. Very few studies explored the efficacy and mechanisms of the action of new chemotherapeutic molecules [29
]. This is the first investigation showing the in vitro effect of tigecycline against Leptospira
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
]. 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 105 Leptospira interrogans
serovar Canicola [35
Considering spirochetes sensu latu, tigecycline showed inhibitory effects on Borrelia
spp. Borrelia burgdorferi
was susceptible to tigecycline at concentrations between 0.006 and 6 µg/mL [36
]. Additionally, for Borrelia bavariensis, B. garinii, B. afzelii, B. spielmanii, B. valaisiana
, and B. lusitaniae
, MIC values between 0.012 and 0.5 µg/mL have been reported [38
]. Moreover, tigecycline MBC values for Borrelia
spp. were between 4 and 16 µg/mL [39
]. Results on the Borrelia
spp. tigecycline effect were similar to those obtained in this investigation against Leptospira
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
]. 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
4. Materials and Methods
4.1. Leptospira spp. Strains
In this investigation, the following references of Leptospira strains were employed: Leptospira interrogans serovar Icterohaemorrhagiae (serogroup Icterohaemorrhagiae, strain Bianchi); L. interrogans serovar Canicola (serogroup Canicola, strain Alarik); L. interrogans serovar Pomona (serogroup Pomona, strain Mezzano); L. kirschneri serovar Grippotyphosa (serogroup Grippotyphosa, strain Moskva V); L. borgpetersenii serovar Tarassovi (serogroup Tarassovi, strain Mitis Johnson); L. interrogans serovar Bratislava (serogroup Australis, strain Riccio 2); L. interrogans serovar Hardjo (serogroup Sejroe, serovar Hardjoprajitno); L. borgpetersenii serovar Ballum (serogroup Ballum, strain Mus 127); L. interrogans serovar Copenhageni (serogroup Icterohaemorrhagiae, strain Wijmberg); L. interrogans serovar Bataviae (serogroup Bataviae, strain Pavia); L. interrogans serovar Zanoni (serogroup Pyrogenes, strain Zanoni); L. borgpetersenii serovar Poi (serogroup Javanica, strain Poi); L. interrogans serovar Lora (serogroup Australis, strain Riccio 37); L. interrogans serovar Autumnalis (serogroup Autumnalis, strain Akiyami A); L. interrogans serovar Hebdomadis (serogroup Hebdomadis, strain Hebdomadis); and Leptospira biflexa serovar Patoc (serogroup Semaranga, strain Patoc I).
Each strain was maintained in pure culture in the Ellinghausen–McCullough–Johnson–Harris (EMJH) medium, sub-cultured, and checked weekly for purity and viability.
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.
4.3. 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
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 × 108
]. 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.