Effective Treatment of Mycobacterium avium subsp. hominissuis and Mycobacterium abscessus Species Infections in Macrophages, Biofilm, and Mice by Using Liposomal Ciprofloxacin

Abstract

Nontuberculous mycobacteria (NTM) affect an increasing number of individuals worldwide. Infection with these organisms is more common in patients with chronic lung conditions, and treatment is challenging. Quinolones, such as ciprofloxacin, have been used to treat patients, but the results have not been encouraging. In this report, we evaluate novel formulations of liposome-encapsulated ciprofloxacin (liposomal ciprofloxacin) in vitro and in vivo Its efficacy against Mycobacterium avium and Mycobacterium abscessus was examined in macrophages, in biofilms, and in vivo using intranasal instillation mouse models. Liposomal ciprofloxacin was significantly more active than free ciprofloxacin against both pathogens in macrophages and biofilms. When evaluated in vivo, treatment with the liposomal ciprofloxacin formulations was associated with significant decreases in the bacterial loads in the lungs of animals infected with M. avium and M. abscessus In summary, topical delivery of liposomal ciprofloxacin in the lung at concentrations greater than those achieved in the serum can be effective in the treatment of NTM, and further evaluation is warranted.

Keywords: Mycobacterium abscessus; Mycobacterium avium; Mycobacterium avium subsp. hominissuis; biofilm; ciprofloxacin; infection; liposome; lung disease; mouse model; treatment.

FIG 1

Activity of 20-μg/ml free ciprofloxacin (FCI 20), liposomal ciprofloxacin (CFI 20), and liposomal nanocrystalline ciprofloxacin (Nano 20) formulations against M. avium subsp. hominissuis in macrophages. (A) M. avium subsp. hominissuis strain MAC101; (B) M. avium subsp. hominissuis strain MAC109. Values are the mean number of CFU and standard deviations (error bars) on days 0 and 4. *, P < 0.05 compared with the initial infecting load (on day 0) in macrophages.

FIG 2

Activity of 200 μg/ml of free ciprofloxacin (FCI 200) and liposomal ciprofloxacin (CFI 200) against Mycobacterium abscessus subsp. abscessus strain MAC101. THP-1 macrophages were infected with M. abscessus (MOI, 2), and after the infection was established (24 h), treatment was started for 4 days. The number of intracellular bacteria (the number of CFU) in the treatment groups after infection (day 4) was compared to the number of intracellular bacteria before treatment (day 0) as well as to the number of bacteria in the untreated buffer control. ELC, empty liposome control with a lipid composition identical to that of CFI at 200 μg/ml. Values are means and standard deviations (error bars). *, P < 0.05 compared to the initial infecting load (on day 0) in macrophages in the untreated buffer control; **, P < 0.02 compared to the initial infecting load (on day 0) in macrophages in the untreated buffer control.

FIG 3

Activity of 100 μg/ml of free ciprofloxacin (FCI 100) and 50 and 100 μg/ml of liposomal ciprofloxacin (CFI 50 and CFI 100, respectively) against M. avium subsp. hominissuis strain A5 (A) and strain MAC104 (B) and M. abscessus strain 105 (C) biofilms. Mature biofilms were established in 96-well plates as described in Materials and Methods. ELC, empty liposome control with a lipid composition identical to the lipid concentration in CFI at 100 μg/ml. CFU values are means and standard deviations (error bars). *, P < 0.05 for the number of bacteria at day 4 versus day 0 before treatment.

FIG 4

Effect of 200 μg/ml of free ciprofloxacin (FCI 200) and liposomal ciprofloxacin (CFI 200) on the formation of a biofilm on HEp-2 cells. HEp-2 cells were cultured to confluence. An antibiotic formulation was added to the culture once. Bacteria were quantified 24 h after the antibiotic was added. CFU values are means and standard deviations (error bars). *, P < 0.05 compared with untreated buffer control at the same time point.

FIG 5

Electron micrographs of M. avium subsp. hominissuis microaggregates. (A) Buffer control added at time zero showing the presence of bacterial microaggregates; (B) free ciprofloxacin (200 μg/ml) treatment added at time zero showing less microaggregate formation than that for the control; (C and D) CFI (200 μg/ml) treatment added at time zero before aggregate formation showing the prevention of aggregation; (E) CFI (200 μg/ml) treatment added at 24 h to already present microaggregates showing an unusual bacterial surface.

FIG 6

Activity of 0.33-, 0.67-, and 1-mg/kg lung doses of free ciprofloxacin (FCI) and liposomal ciprofloxacin (liposomal ciprofloxacin [CFI 0.33, CFI 0.67, and CFI 1.0] and Linhaliq [LIN 0.33, 0.67, and 1.0]) against MAC strain MAC104 in mice over 3 weeks in lung (A) and spleen (B). Six-week-old C57BL/6 mice were used. Mice were infected and treated by intranasal instillation. ELC, empty liposome control with a lipid composition identical to that of CFI at 1 mg/kg. CFU values are means and standard deviations (error bars). *, P < 0.05 compared with saline or empty liposome control at week 3; †, P < 0.05 compared with 1-mg/kg free ciprofloxacin at week 3.

FIG 7

Efficacy of 1-mg/kg free ciprofloxacin (FCI 1.0) and liposomal ciprofloxacin (liposomal ciprofloxacin [CFI 1.0] and Linhaliq [LIN 1.0]) against MAC strain MAC104 in mice over 3 and 6 weeks. Six-week-old C57BL/6 mice were used and were infected and treated by intranasal instillation. ELC, empty liposome control with a lipid concentration identical to that of CFI at 1.0 mg/kg. CFU values are means and standard deviations (error bars). *, P < 0.05 compared with saline at week 1; †, P < 0.05 compared with the empty liposome control after infection (day 4) in the same week 1; ‡, P < 0.05 for the parameter at week 6 compared to week 3.

FIG 8

Mean number of CFU of M. abscessus strain 101 in lung (A) and spleen (B) in two studies in C57BL/6 beige bj/bj mice. Mice were infected with 1 × 10⁷ CFU by intranasal instillation, observed for the stated number of days, and then harvested, and the lungs and spleen were plated onto Middlebrook 7H10 agar plates.

FIG 9

Efficacy of 1 mg/kg of free ciprofloxacin (FCI 1.0) and liposomal ciprofloxacin (liposomal ciprofloxacin [CFI 1.0] and Linhaliq [LIN 1.0]) in the treatment of M. abscessus strain 101 infection in the lung (A) and spleen (B). C57BL/6 beige bj/bj mice were infected with (5.4 ± 0.3) × 10⁷ CFU in 0.5 ml of HBSS by intranasal instillation; treatment was also by intranasal instillation. ELC, empty liposome control with a lipid composition identical to that in CFI at 1 mg/kg. Values are means and standard deviations (error bars). *, P < 0.05 compared with saline, the empty liposome control, and free ciprofloxacin in the same week; †, P < 0.05 for the parameter at week 6 compared to week 3.