A BALB/c mouse model of Mycobacterium abscessus lung infection based on once-weekly cyclophosphamide administration

Abstract

Mycobacterium abscessus is a fast-growing non-tuberculous mycobacterium that can cause chronic lung disease leading to rapid decline in lung function. There are no FDA-approved therapies for this disease. To support the development of new treatments, an animal model of M. abscessus lung infection that is simple to implement and requires minimal resources is crucial to encourage broad adoption. We present a mouse model using the immunocompetent BALB/c strain, which is both widely available and cost effective. Since BALB/c mice naturally clear M. abscessus infections, immunosuppression is necessary to sustain bacterial growth in the lungs. Once-weekly intraperitoneal injections of the immunosuppressant cyclophosphamide at 250 mg/kg successfully induced proliferation of M. abscessus during the acute phase, followed by stabilization characteristic of chronic infection. This model demonstrated the efficacy of imipenem - an antibiotic commonly used in clinical settings - by significantly reducing bacterial burdens, mirroring their effects in human cases. However, clofazimine, which is also used to treat this disease, was bacteriostatic. This cost-effective and accessible mouse model is suitable for diverse laboratory environments and provides a valuable tool for preclinical evaluation of treatments for M. abscessus lung disease.

Keywords: Mycobacterium abscessus; Mycobacteroides abscessus; Lung infection; Mouse model.

Fig. 1.

Schematic overview of Study 1 and plot showing mean Mab burden in the lungs of BALB/c mice. Top: Schematic of the experimental set-up for Study 1. All mice received a single 200 mg/kg bolus of cyclophosphamide as a loading dose 3 days before infection (Day -3) and a 100 mg/kg bolus 2 h prior infection with M. abscessus (Mab) (Week 0). After infection, mice in Group A (Scheme A; red) received a weekly dose of 100 mg/kg bolus cyclophosphamide for 3 weeks (Week 1 to Week 4), while mice in Group B (Scheme B; blue) received no further cyclophosphamide treatment. n=5. Bottom: Plotted is the Mab burden in the lungs of Group A (red) and Group B (blue) mice as the mean colony-forming unit (CFU)/lung (±s.d.) over 4 weeks. In Group A (red graph), receiving a weekly dose of 100 mg/kg cyclophosphamide, lung Mab burden increased by 0.3 log₁₀ at the end of Week 1 but declined thereafter, leading to a net reduction of 2.0 log₁₀ over the 4-week-long examination period, resulting in a paucibacillary infection. Over the same period, control Group B (blue graph) showed a similar total net reduction in lung Mab burden of 1.7 log₁₀.

Fig. 2.

Schematic overview of Study 2 and plot showing the mean Mab burden in the lungs of BALB/c mice. Top: Schematic of the experimental set-up for Study 2. All mice received a loading dose of 200 mg/kg cyclophosphamide 3 days before infection (Day -3) and again one dose 2 h before infection with M. abscessus (Mab) (Week 0). After infection, mice in Group C received 200 mg/kg cyclophosphamide twice weekly, i.e. a total of 400 mg/kg per week, with individual doses 3.5 days apart. Mice in Group D received 200 mg/kg cyclophosphamide once weekly. n=5. Mean CFU/lung±s.d. is plotted. Bottom: Plotted is the Mab burden in the lungs of Group C (red) and D (blue) mice as the mean colony-forming unit (CFU)/lung (±s.d.) over 4 weeks. In Group C (red graph), lung Mab burden increased steadily each week for 3 weeks, followed by a decrease of 0.5 log₁₀ CFU in Week 4, resulting in a net increase of 3.2 log₁₀ CFU over the 4-week period. In Group D (blue graph), increased lung Mab burden at the end of Week 1 was followed by a decrease in the following week with a net increase of 0.5 log₁₀ CFU over the 4-week period.

Fig. 3.

Schematic overview of Study 3 and plot showing the mean Mab burden in the lungs of BALB/c mice. Top: Schematic of the experimental set-up for Study 3. All mice received a single loading dose of 250 mg/kg cyclophosphamide 3 days before infection (Day -3) and one dose 2 h before infection with M. abscessus (Mab) (Week 0), followed by one dose once weekly for three subsequent weeks (Week 1 to Week 3). After the end of three weeks following infection, cyclophosphamide treatment was discontinued in Group F (blue). Mice in Group E (red) continued to receive a once weekly bolus of 250 mg/kg cyclophosphamide intraperitoneally. n=5. Mean CFU/lung±s.d. is plotted. After the end of three weeks following infection (Week 4), injection of cyclophosphamide was replaced by injection of PBS (control) in Group F (blue). Group E (red) continued to receive a once-weekly bolus of 250 mg/kg cyclophosphamide intraperitoneally until Week 8. n=5. Bottom: Plotted is the Mab burden in the lungs of Group E (red) and F (blue) mice as the mean colony-forming unit (CFU)/lung (±s.d.) over 8 weeks.

Fig. 4.

Schematic overview of Study 4 and plot showing the mean Mab burden in the lungs of BALB/c mice. Top: Schematic of the experimental set-up for Study 4. As for Study 3, all mice received a single loading dose of 250 mg/kg cyclophosphamide 3 days before infection (Day -3) and one dose 2 h before infection with M. abscessus (Mab) (Week 0), followed by one dose once weekly for three subsequent weeks (Week 1 to Week 3). After the end of three weeks following infection (Week 4), injection of cyclophosphamide was replaced by injection of PBS (control) in Group F (blue) Group E (red) continued to receive a once-weekly bolus of 250 mg/kg cyclophosphamide intraperitoneally until Week 10. At the beginning of week 4, Group G (green) received imipenem (100 mg/kg per dose) two doses per day (8–12 h apart), 7 days a week. n=5. Bottom: Plotted is the Mab burden in the lungs of Group E (red), G (green) and F (blue) as the mean colony-forming unit (CFU)/lung (±s.d.) over 10 weeks. For Group F, CFU counts were 0, 3, 0, 49 and 0. For mice with undetectable CFUs, a log₁₀ CFU value of 0 was assigned. As a result, the mean log₁₀ CFU of Mab burden is <1.

Fig. 5.

Schematic overview of Study 5 and plot showing the mean Mab burden in the lungs of BALB/c mice. Top: Schematic of the experimental set-up for Study 5. All mice received a single loading dose of 250 mg/kg cyclophosphamide 3 days before infection (Day -3) and one dose 2 h before infection with M. abscessus (Mab) (Week 0), followed by one dose once weekly for 7 subsequent weeks (Week 1 to Week 7). Groups E (red) and H (purple) continued to receive a once weekly bolus of 250 mg/kg cyclophosphamide intraperitoneally until Week 7. Group H (purple) received once daily oral 25 mg/kg clofazimine from start of week 4 until week 8. n=5. Bottom: Plotted is the Mab burden in the lungs of Group E (red) and H (purple) mice as the mean colony-forming unit (CFU)/lung (±s.d.) over 8 weeks.