(M0930-01-02) Exploring the Impact of Iron Availability on Morphology and Antibiotic Resistance in Mycobacterium Species

Purpose: Iron is essential for the survival and pathogenesis of Mycobacterium tuberculosis (Mtb), the bacterium responsible for tuberculosis (TB). It plays a crucial role in cellular respiration, DNA synthesis, and metabolic function. Mtb adapts to iron availability by producing siderophores to scavenge iron, using transporters to import it, and storing excess iron in proteins like bacterioferritin and ferritin. These strategies enable Mtb to thrive in iron-limited environments such as within the host’s immune cells. Iron availability influences Mtb's virulence, impacting its ability to persist inside host cells and evade immune responses. It also affects Mtb's growth, cholesterol metabolism, and antibiotic tolerance, with iron deficiencies often impairing these functions. Restoring iron levels can reverse these defects, highlighting the importance of iron in Mtb’s pathogenicity. Our laboratory is developing a novel iron delivery system using Metal-Organic Frameworks (MOFs) like MIL-100 and MIL-88A. These MOFs can release iron in a controlled manner, potentially enhancing bacterial persistence or causing iron overload that leads to bacterial death. This approach aims to manipulate Mtb’s iron homeostasis, offering new therapeutic avenues for combating tuberculosis by either promoting or disrupting iron balance within the pathogen.

Methods: Oppressive environments are believed to induce the emergence of rough and smooth (R/S) morphologies in Mycobacteria. This study investigates the relationship between R/S morphology and iron availability in both fast-growing Mycobacterium smegmatis and slow-growing Mycobacterium avium. The emergence of R/S colonies in M. smegmatis was assessed after exposure to Metal-Organic Frameworks (MOFs) and free iron (FeCl₃). Additionally, individual R/S colonies were exposed to MOFs, FeCl₃, and the iron chelator Deferoxamine (DFO) at various concentrations for 24 hours. After exposure, colonies were grown on 7H10 agar plates for one week, and colony counts were recorded. To investigate nutrient effects, metal-deprived agar and media were prepared. For antibiotic susceptibility testing, Isoniazid (INH) and Rifampicin (RIF) were used in minimum inhibitory concentration (MIC) assays to assess any changes in antibiotic resistance. Morphological changes in R/S colonies were observed on 7H10 agar plates. This study aims to clarify how varying iron availability influences R/S colony formation and antibiotic susceptibility in Mycobacteria. Understanding these relationships could provide valuable insights into how Mycobacteria adapt to hostile environments and potentially enhance their resistance to treatment.

Results: In Mycobacterium species, the wildtype morphology typically differentiates into a dominant rough morphology. When exposed to Metal-Organic Frameworks (MOFs) and free iron, the colony-forming units (CFU) decrease compared to the control. When examining the response of rough and smooth colonies to MOFs, smooth colonies show a decreased CFU with increasing MOF concentration. In contrast, rough morphology exhibits an increase in CFU at low MOF concentrations, which then decreases as the concentration rises. Different nutrient environments also influence morphology. Bacteria grown in nutrient-restricted conditions tend to exhibit a more transparent appearance than the wildtype. Furthermore, under nutrient deprivation and exposure to Isoniazid (INH), bacteria display a completely transparent morphology, indicating a severe stress response. The addition of free iron (10 µg/ml) alongside INH results in improved growth of M. avium despite antibiotic pressure, in contrast to the control without antibiotics. This suggests that iron supplementation can partially alleviate the effects of antibiotic stress. These findings highlight how both iron availability and nutrient stress can influence the morphology and antibiotic susceptibility of Mycobacterium species, providing insights into their adaptive mechanisms under hostile conditions.

Conclusion: In conclusion, this study underscores the critical role of iron in the survival and pathogenicity of Mycobacterium species, particularly Mycobacterium tuberculosis and Mycobacterium avium. Iron availability influences bacterial morphology, growth, and antibiotic resistance, with the emergence of rough and smooth (R/S) morphologies serving as an adaptive response to environmental stressors. Our results reveal that exposure to Metal-Organic Frameworks (MOFs) and free iron significantly impacts the colony-forming units (CFU) of M. smegmatis and M. avium, with varying responses observed between rough and smooth colonies. Additionally, iron supplementation in the presence of antibiotic pressure can enhance bacterial growth, highlighting iron's potential role in mitigating antibiotic stress. Nutrient limitation further exacerbates these morphological changes, with nutrient-deprived conditions inducing more transparent, stress-induced morphologies. These findings provide valuable insights into how Mycobacterium species adapt to hostile environments, including nutrient scarcity and antibiotic treatment. By manipulating iron availability through novel approaches like MOFs, we may gain new therapeutic strategies to combat tuberculosis by either promoting or disrupting the pathogen's iron homeostasis. Understanding the complex relationship between iron, morphology, and antibiotic resistance offers a promising avenue for improving treatment outcomes and addressing the challenges posed by drug-resistant Mycobacterium infections.

Acknowledgements: 1RO1AI152109-01, National Institute of Allergy And Infectious Disease, Pharmaceutical Science Department

Bar graph of colonies counted after MOF or free iron (FeCl3) exposure. Data are given as means ± SD

Iron concentration effect on bacterial growth

Rough or smooth colony exposure to iron excess (FeCl3) or iron deprivation by iron chelator deferoxamine (DFO). Data are given as means ± SD