Impact of azithromycin resistance mutations on the virulence and fitness of Chlamydia caviae in guinea pigs

Abstract

Azithromycin (AZM) is a major drug used in the treatment and prophylaxis of infections caused by Chlamydia, yet no significant clinical resistance has been reported for these obligate intracellular bacteria. Nevertheless, spontaneous AZM resistance (Azm(r)) arose in vitro at frequencies ranging from 3 x 10(-8) to 8 x 10(-10) for clonal isolates of Chlamydia caviae, which is a natural pathogen of guinea pigs. Sequencing of the unique 23S rRNA gene copy in 44 independent Azm(r) isolates identified single mutations at position A(2058) or A(2059) (Escherichia coli numbering system). While SP(6)AZ(1) (A(2058)C) and SP(6)AZ(2) (A(2059)C) Azm(r) mutants showed growth defects in cell culture and were less pathogenic in the guinea pig ocular infection model than in the parent SP(6), the three isogenic C. caviae isolates grew equally well in the animal. On the other hand, coinoculation of the C. caviae parent strain with one of the Azm(r) strains was detrimental for the mutant strain. This apparent lack of association between pathology and bacterial load in vivo showed that virulence of the two Azm(r) mutants of C. caviae was attenuated. While chlamydial growth in vitro reflects the ability of the bacteria to multiply in permissive cells, survival in the host is a balance between cellular multiplication and clearance by the host immune system. The obligate intracellular nature of Chlamydia may therefore limit emergence of resistance in vivo due to the strength of the immune response induced by the wild-type antibiotic-sensitive bacteria at the time of antibiotic treatment.

FIG. 1.

Impact of AZM resistance mutations on the growth of C. caviae SP₆ variants in mouse fibroblast L2 cells. Confluent mouse fibroblasts in 60-mm dishes were infected with wild-type C. caviae SP₆ and two isogenic mutants with mutations in the 23S rRNA gene, SP₆AZ₁ (A₂₀₅₈C) and SP₆AZ₂ (A₂₀₅₉C). The growth medium from each dish (i.e., culture supernatant) and then the infected monolayers were collected at regular time intervals. Infectious particles were harvested into 400 μl SPG. IFU were enumerated after infection onto McCoy cells and immunodetection of the chlamydial inclusions at 24 h p.i. The total number of infectious particles was determined by adding both titers, i.e., those of the supernatant and the infected monolayer, at the respective time point. Data are expressed as means ± standard deviations of results from triplicate determinations and are related to the number of IFU added to the monolayer.

FIG. 2.

Impact of AZM resistance mutations on the virulence of C. caviae SP₆ variants in the natural host. Ten thousand IFU from the three isogenic C. caviae isolates, SP₆, SP₆AZ₁ (A₂₀₅₈C), and SP₆AZ₂ (A₂₀₅₉C), were separately inoculated in both eyes in five animals per sample. Two independent experiments were performed and showed the same trend. The mean course of infection for one set is presented. (A) Recovery of infectious particles. Two swabs were collected every third day from the second eye. IFU were enumerated after infection onto McCoy cells from one swab, and the other was stored at −80°C. The data points represent the mean numbers of IFU for the 5 animals in the group. There were no significant differences among the 3 groups as determined by a 2-factor ANOVA with repeated measures. (B) Conjunctival pathology. Conjunctival pathology was scored every day for each eye on a scale from 0 to 4. The scores represent the means of results from 10 eyes. The scores for both eyes of an animal were always consistent, i.e., different by no more than 1 number. The pathology of animals infected with both SP₆AZ₁ and SP₆AZ₂ was significantly different from that obtained with the parent strain (P < 0.001), as determined by a 2-factor ANOVA with repeated measures.

FIG. 3.

Conjunctival pathology observed after single infection or coinoculation of wild-type C. caviae SP6 and the two 23S rRNA mutants, SP₆AZ₁ (A₂₀₅₈C) and SP₆AZ₂ (A₂₀₅₉C), in the natural host. Ten thousand IFU from the wild type and one Azm^r isogenic mutant of C. caviae were coinoculated in both eyes of five animals per sample. Conjunctival pathology was scored as described in the legend to Fig. 2. Results from the coinfection experiments are shown with dotted lines, while 12 results from the single infections (from Fig. 2B) are shown with solid lines. (A) The scores of the coinoculation group (P < 0.008) and the SP₆AZ₁-alone group (P < 0.001) were significantly different from that of the SP₆-alone group as determined by a 2-factor ANOVA with repeated measures; however, the score of the SP₆AZ₁-alone group was significantly lower than that of the coinoculation group (P < 0.016). (B) The score of the coinoculation group (P < 0.008) was not significantly different from that of the SP₆-alone group as determined by a 2-factor ANOVA with repeated measures. However, the score of the SP₆AZ₂-alone group (P < 0.001) was significantly lower than those of both the coinoculation group (P < 0.001) and the SP₆-alone group (P < 0.001).

FIG. 4.

Competition between wild-type C. caviae SP₆ and the two 23S rRNA mutants, SP₆AZ₁ (A₂₀₅₈C) and SP₆AZ₂ (A₂₀₅₉C), in the natural host. Coinoculation was performed as described in the legend to Fig. 3. Two swabs were collected every third day from one eye. IFU were enumerated after inoculation onto McCoy cells with (AZM IFU) and without (total IFU) AZM. The number of wild-type IFU was calculated as the total number of IFU minus the number of Azm^r IFU. Results from the coinfection experiments are shown with dotted lines, while results from the single infections (from Fig. 2A) are shown with solid lines. (A) The level of SP₆AZ₁ in the coinoculation group was significantly lower than the levels of SP₆AZ₁ alone (P < 0.001), SP₆ alone (P < 0.001), and SP₆ in the coinoculation group (P < 0.001) as determined by a 2-factor ANOVA with repeated measures. The level of SP₆AZ₁ alone was not significantly lower than that of SP₆ alone, but the level of SP₆ in the coinoculation group was significantly different from that of SP₆ alone (P < 0.001). (B) The level of SP₆AZ₂ in the coinoculation group was significantly lower than the levels of SP₆AZ₂ alone (P < 0.001), SP₆ alone (P < 0.001), and SP₆ in the coinoculation group (P < 0.001) as determined by a 2-factor ANOVA with repeated measures. The level of SP₆AZ₂ alone was significantly lower than that of SP₆ alone (P < 0.02), and the level of SP₆ in the coinoculation group was significantly different from that of SP₆ alone (P < 0.001).