Assessment of Antimicrobial Therapy in Eradicating Chlamydia muridarum in Research Mice: Immune Status and Its Impact on Outcomes

Abstract

Chlamydia muridarum (Cm) is a moderately prevalent, gram-negative, intracellular bacterium that affects laboratory mice, causing subclinical to severe disease, depending on the host's immune status. The effectiveness of various antibiotic regimens aimed at eradicating Cm in both immunodeficient and immunocompetent laboratory mice was evaluated. NSG mice were cohoused with Cm-shedding BALB/cJ mice for 14 d to simulate natural exposure. Four groups of 8 infected NSG mice were treated for 7 d with either 0.08% sulfamethoxazole and 0.016% trimethoprim (TMS) in water, 0.0625% doxycycline in feed, 0.124%/0.025% TMS in feed, or 0.12% amoxicillin in feed. A control group was provided standard water and feed. The impact of treatment on gastrointestinal microbiota (GM) was investigated using next-generation shotgun sequencing on the last day of treatment. TMS and amoxicillin had negligible effects on GM, while doxycycline had the largest effect. All antibiotic-treated NSG mice exhibited clinical disease, including dehydration, hunched posture, greater than 20% weight loss, and dyspnea, leading to euthanasia 21 to 40 d posttreatment (32.6 ± 4.2 d; mean ± SD). Untreated controls were euthanized 14 to 33 d postexposure (23.75 ± 5.9 d). All mice were fecal PCR positive for Cm at euthanasia. Histologic evaluation revealed multifocal histiocytic and neutrophilic bronchointerstitial pneumonia and/or bronchiolitis featuring prominent intralesional chlamydial inclusion bodies in all mice. Subsequently, groups of 8 C57BL/6J, BALB/cJ, NOD.SCID, and NSG mice infected with Cm were treated with 0.124%/0.025% TMS in feed for 7 (BALB/cJ and C57BL/6J) or 21 d (NSG and NOD.SCID). All immunocompetent and NOD.SCID mice were negative for Cm by PCR 14 d posttreatment, remained clinically normal, and had no evidence of Cm infection at necropsy, and all NSG mice remained Cm positive and were euthanized. While these findings highlight the difficulties in eradicating Cm from highly immunodeficient mice, eradication of Cm from immunocompetent or moderately immunocompromised mice with antibiotics is feasible.

Keywords: AB, aberrant body; B6, C57BL/6J; C, BALB/cJ; Cm, Chlamydia muridarum; DPT, days posttreatment; EB, elementary body; GEM, genetically engineered mouse; GM, gastrointestinal microbiota; IB, inclusion body; MOMP, major outer membrane protein; NOD.SCID, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ; NSG, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ; RB, reticulate body; SW, Tac:SW; TMS, trimethoprim and sulfamethoxazole.

Figure 1.

Cm-infected NSG mouse Kaplan-Meier survival curve for untreated and 7-d treatment groups. Day 0 denotes the final day of antibiotic administration.

Figure 2.

PCoA of samples collected from NSG mice (n = 20) after randomization but before cohousing with C mice using Bray-Curtis similarity. Colored datapoints denote untreated mice destined for a specific treatment. Manhattan distance was not included due to similarity of the plot to Bray-Curtis similarity.

Figure 3.

α-Diversity and PCoA of samples collected from NSG mice (n = 20) after randomization and cohousing but before antibiotic treatment using (A) Shannon index, (B) Simpson index, and (C) Bray-Curtis similarity. Manhattan distance was not included due to similarity of the plot to Bray-Curtis similarity. Lines indicate significant (P < 0.05) time-dependent differences as determined via paired t test.

Figure 4.

α-Diversity and PCoA plots for samples collected from each group (n = 4) before (pre) and on the last day of (post) antibiotic treatment using (A) Shannon index, (B) Simpson index, (C) Manhattan distance, and (D) Bray-Curtis similarity. (C) and (D) are shown on the next page. Red lines indicate significant (P < 0.05) time- and antibiotic-dependent differences as determined via paired t test or Wilcoxon-matched pairs signed-rank tests. Abx, antibiotics.

Figure 4.

α-Diversity and PCoA plots for samples collected from each group (n = 4) before (pre) and on the last day of (post) antibiotic treatment using (A) Shannon index, (B) Simpson index, (C) Manhattan distance, and (D) Bray-Curtis similarity. (C) and (D) are shown on the next page. Red lines indicate significant (P < 0.05) time- and antibiotic-dependent differences as determined via paired t test or Wilcoxon-matched pairs signed-rank tests. Abx, antibiotics.