Cethromycin-mediated protection against the plague pathogen Yersinia pestis in a rat model of infection and comparison with levofloxacin

Abstract

The Gram-negative plague bacterium, Yersinia pestis, has historically been regarded as one of the deadliest pathogens known to mankind, having caused three major pandemics. After being transmitted by the bite of an infected flea arthropod vector, Y. pestis can cause three forms of human plague: bubonic, septicemic, and pneumonic, with the latter two having very high mortality rates. With increased threats of bioterrorism, it is likely that a multidrug-resistant Y. pestis strain would be employed, and, as such, conventional antibiotics typically used to treat Y. pestis (e.g., streptomycin, tetracycline, and gentamicin) would be ineffective. In this study, cethromycin (a ketolide antibiotic which inhibits bacterial protein synthesis and is currently in clinical trials for respiratory tract infections) was evaluated for antiplague activity in a rat model of pneumonic infection and compared with levofloxacin, which operates via inhibition of bacterial topoisomerase and DNA gyrase. Following a respiratory challenge of 24 to 30 times the 50% lethal dose of the highly virulent Y. pestis CO92 strain, 70 mg of cethromycin per kg of body weight (orally administered twice daily 24 h postinfection for a period of 7 days) provided complete protection to animals against mortality without any toxic effects. Further, no detectable plague bacilli were cultured from infected animals' blood and spleens following cethromycin treatment. The antibiotic was most effective when administered to rats 24 h postinfection, as the animals succumbed to infection if treatment was further delayed. All cethromycin-treated survivors tolerated 2 subsequent exposures to even higher lethal Y. pestis doses without further antibiotic treatment, which was related, in part, to the development of specific antibodies to the capsular and low-calcium-response V antigens of Y. pestis. These data demonstrate that cethromycin is a potent antiplague drug that can be used to treat pneumonic plague.

Fig. 1.

Evaluation of various doses of levofloxacin (Levo) in a pneumonic rat model of infection. Groups of 6 rats were exposed to a 12 LD₅₀ of Y. pestis CO92 and administered levofloxacin at the indicated doses. Additionally, 9 rats were infected and received PBS instead of levofloxacin as a negative control. The duration of the drug treatment is indicated by the red bar. The animals in the levofloxacin-treated groups were rechallenged and monitored for lethality. P values indicate statistical significance.

Fig. 2.

Evaluation of dose-response effect of cethromycin in a pneumonic rat model of infection. Groups of 9 rats were exposed to a 25 LD₅₀ of Y. pestis CO92 and administered cethromycin at the indicated doses. A negative control consisted of rats administered PBS instead of cethromycin (Cethro), and as a positive control rats received 20 mg/kg/day of levofloxacin (Levo) either orally or intraperitoneally. Drug treatments were administered for either 7 or 14 days, and administration lengths are indicated by red and blue bars. P values indicate statistical significance.

Fig. 3.

Evaluation of higher oral doses of cethromycin in a pneumonic rat model of infection and subsequent protection from 2 Y. pestis rechallenges. Groups of rats were exposed to a 30 LD₅₀ of Y. pestis CO92 and administered cethromycin (Cethro) at the indicated doses. As a negative control, rats were administered PBS instead of cethromycin, while as a positive control, rats received 20 mg/kg/day of levofloxacin (Levo) orally. Drug treatments were administered for 6, 10, or 14 days, and administration lengths are indicated by different colored bars. Survivors were rechallenged twice following the first Y. pestis infection. As toxicity controls, uninfected rats were administered either 120 mg/kg or 140 mg/kg/day for 14 days. P values indicate statistical significance.

Fig. 4.

Evaluation of 70 mg/kg b.i.d. of cethromycin in a pneumonic rat model of infection. Groups of rats were exposed to a 24 LD₅₀ of Y. pestis CO92 and administered cethromycin (Cethro) at 70 mg/kg b.i.d., resulting in a total daily dose of 140 mg/kg. As a negative control, rats were administered PBS instead of cethromycin. A total daily dose of 140 mg/kg for 7 days was also carried out to evaluate any drug-induced animal toxicity.

Fig. 5.

Evaluation of lower oral b.i.d. doses of cethromycin in a pneumonic rat model of infection. Groups 9 rats were exposed to a 30 LD₅₀ of Y. pestis CO92 and administered cethromycin (Cethro) at the indicated doses. As a negative control, rats were administered PBS instead of cethromycin. The duration of the drug treatment is indicated by the red bar. P values indicate statistical significance.

Fig. 6.

Evaluation of delayed administration of various doses of levofloxacin in a pneumonic rat model of infection. Groups of rats exposed to a 12 LD₅₀ of Y. pestis CO92 and 24 to 48 h following Y. pestis infection were i.p. administered levofloxacin (Levo) at the indicated doses. As a negative control, 9 rats were infected and administered PBS. The solid black vertical line indicates rechallenging of the surviving animals. A group of 9 naïve rats was infected and served as rechallenge control. P values indicate statistical significance.

Fig. 7.

Evaluation of delayed administration of cethromycin in a pneumonic rat model of infection. Groups of rats were exposed to a 30 LD₅₀ of Y. pestis CO92 and administered cethromycin (Cethro) at 70 mg/kg/b.i.d. at 24 to 60 h following Y. pestis infection. As a negative control, rats were infected and administered water. The solid black vertical line indicates when the surviving animals were rechallenged with Y. pestis. P values indicate statistical significance.

Fig. 8.

Y. pestis-infected and cethromycin-rescued rats developed antibodies to F1 and LcrV. Significant antibody titers to both F1 and LcrV were developed in rats after the first Y. pestis infection (top) and after rechallenge (bottom) based on ELISA. We used sera from infected rats (those described above for Fig. 7) that received cethromycin 24 h postinfection and were rechallenged (n = 9). The ELISA titers (shown as average means ± standard deviations [SD]) were statistically significant compared to naïve rat sera. The inset in this figure shows that rechallenge sera contained specific antibodies to LcrV (lane 1; 37 to 38 kDa) and F1 (lane 2; 15 to 16 kDa) based on Western blot analysis.