In Vitro Susceptibility of Rickettsia Species to Eravacycline, Omadacycline, and Tigecycline

LETTER

Members of the genus Rickettsia are obligately intracellular vector-borne bacteria that cause acute febrile illness (1). Doxycycline is the drug of choice for all rickettsioses (2). To circumvent modes of tetracycline resistance, such as the emergence of efflux pumps and ribosomal protection produced by more typical bacteria (e.g., Staphylococcus aureus and members of the Enterobacteriaceae), new tetracycline-like derivatives—tigecycline, omadacycline, and eravacycline—have been developed (3, 4). There are no known naturally occurring tetracycline-resistant strains of Rickettsia. Considering the limited antibiotic exposure of their primary reservoirs (i.e., arthropods) and evolution through intense genome reduction (minimizing their genetic plasticity), it is unlikely that spontaneous tetracycline resistance of Rickettsia would occur. Considering the activity of these tetracycline-like agents against resistant but more typical pathogens, their empirical use is likely to expand. Thus, their usefulness against rickettsiae, the cause of empirically treated undifferentiated febrile illnesses, should be sought. Here, we describe the in vitro effectiveness of eravacycline, omadacycline, and tigecycline against pathogenic rickettsiae.

Tested rickettsiae were chosen to represent species from a broad geographic range (Table 1). Eravacycline, omadacycline, tigecycline, and doxycycline were reconstituted and stored according to manufacturers’ labels. For each Rickettsia sp., and for each antibiotic, a cell culture assay was prepared using six-well plates with confluent Vero cells as previously described (5). Assay medium was prepared with each antibiotic at concentrations of 4 μg/ml and down to 0.06 μg/ml, following serial 2-fold dilutions. Antibiotic-free wells were used as a control. Fresh medium with antibiotics was prepared and changed at 48 h. Monolayers were collected on day 0 and 96 h after initial rickettsial inoculation, DNA was extracted from each well, and quantitative PCR was performed. Each drug concentration and all controls were performed in duplicate wells for each assay. Assays were performed three times. We defined the MIC as the lowest concentration of antibiotic which inhibited the growth of organisms to a level equal to or less than the quantity of bacteria on day 0. The MIC₅₀ and MIC₉₀ were determined by finding the MIC effective against 5 and 9, respectively, of the 10 rickettsial species tested.

TABLE 1.

Activities of eravacycline, omadacycline, and tigecycline against 10 rickettsial species

Species or straina	MIC (μg/ml)
	Eravacycline	Omadacycline	Tigecycline	Doxycycline
Rickettsia africae	≤0.06	0.5	0.5	≤0.06
Rickettsia australis	≤0.06	0.5	1.0	≤0.06
Rickettsia conorii India tick typhus strain	≤0.06	0.5	0.5	≤0.06
Rickettsia conorii Malish 7 strain	≤0.06	1.0	1.0	≤0.06
Rickettsia honei	≤0.06	1.0	0.5	0.13
Rickettsia parkeri	≤0.06	0.5	0.5	0.13
Rickettsia rickettsii	≤0.06	1.0	0.5b	0.13
Rickettsia sibirica	≤0.06	1.0	1.0	≤0.06
Rickettsia slovaca	≤0.06	2.0	1.0	0.13
Rickettsia typhi	≤0.06	0.5	1.0	≤0.06
MIC50c	≤0.06	0.5	0.5	≤0.06
MIC90c	≤0.06	1.0	1.0	0.13

^aThe Rickettsia strains tested include representatives from the three clades with pathogenic members. Most are of the spotted fever group. Rickettsia typhi and R. australis are members of the typhus and transitional groups, respectively.

^bThe susceptibility of R. rickettsii to tigecycline was demonstrated previously (5).

^cMIC₅₀ and MIC₉₀ for the listed species.

The MICs of these antimicrobials for the tested rickettsial species are shown in Table 1. The MIC of eravacycline for each species was ≤0.06 μg/ml. The MIC for omadacycline ranged from 0.5 to 2 μg/ml. Its MIC₅₀ and MIC₉₀ were 0.5 μg/ml and 1.0 μg/ml, respectively. The MIC was 2 μg/ml against only one species, Rickettsia slovaca, the cause of a mild rickettsiosis. The MIC of tigecycline was 0.5 to 1.0 μg/ml, with an MIC₅₀ and MIC₉₀ of 0.5 μg/ml and 1.0 μg/ml, respectively. Similar variations in the in vitro susceptibility of rickettsiae to various antibiotics were documented previously (6). For each rickettsial isolate, doxycycline was used as a control to verify the validity of each assay. Doxycycline exhibited MICs of 0.06 to 0.13 μg/ml, consistent with its known activity (6, 7).

Drug susceptibility assays for rickettsiae are not routinely used, nor are they as well validated as they are for more typical bacteria (2). The use of quantitative PCR to determine the susceptibility of Rickettsia spp., as used here, was established previously and produces results similar to those of other methods (7). Although there are numerous named species within the genus Rickettsia, the degree of genetic difference used to classify species in this genus is a fraction of that used for other bacteria. Indeed, contemporary methods comparing whole genomes have proposed that the plethora of species could be greatly condensed (8). Furthermore, signs and symptoms of rickettsioses have marked overlap (1). For these reasons, the MIC₅₀ and MIC₉₀ were calculated using different species or strains, rather than using multiple isolates of a single species. Although each rickettsia tested is of clinical significance, these individual isolates have been passaged numerous times in Vero cells. It is possible that these cell culture-adapted isolates could have different susceptibility than those in vivo.

The maximum concentrations in serum (C_max) of eravacycline, omadacycline, and tigecycline are 1.83 μg/ml, 2.12 μg/ml, and 0.98 μg/ml, respectively (9). Considering their pharmacokinetics, their ability to concentrate within cells (9–12), and their aforementioned in vitro activity against rickettsiae, these agents may be effective in the treatment of a variety of rickettsioses. While eravacycline and tigecycline are limited to intravenous use, omadacycline is available in both oral and intravenous formulations. In some Latin American countries, where Rocky Mountain spotted fever (the most severe rickettsiosis) is endemic, intravenous formulations of doxycycline are often not available. Although more expensive than doxycycline, other parenteral agents (such as tigecycline) are more readily accessible (5). The further study of these drugs in animal models of rickettsial diseases should be pursued.