Treatment of bacterial biothreat agents with a novel purified bioactive lactoferrin affects both growth and biofilm formation

Abstract

Lactoferrin is known to exhibit broad spectrum activity against a multitude of bacteria, fungi, and viruses due to its multi-functional mode of action. Recently, Lactea Therapeutics and its affiliates have developed a novel, patent-pending technology to purify naturally derived bovine lactoferrin (Lactea Lf) for use as a medical countermeasure that was not previously available. To assess the efficacy of Lactea Lf against biothreat pathogens, we performed biofilm inhibition assays and generated dose-response curves against Burkholderia pseudomallei, Burkholderia mallei, and Francisella tularensis for proof-of-principle studies. Here, we show that Lactea Lf can significantly inhibit biofilm and decrease the overall growth in a dose dependent manner for all Burkholderia species tested. Of note, Lactea Lf was found to completely inhibit biofilm formation by virulent B. pseudomallei without observing complete growth inhibition. The growth of F. tularensis was also significantly inhibited when cultured in the presence of Lactea Lf and appeared more sensitive to treatment when compared to B. pseudomallei. Based on these results, a pneumonic infection model using the F. tularensis LVS strain was performed prophylactically administering Lactea Lf and continuing treatment post challenge. No protection was observed in this model which prompted biodistribution studies using fluorescent tagged Lactea Lf. These experiments revealed that therapeutic material was mainly confined to the NALT region following intranasal delivery and then quickly dispersed or inactivated suggesting that future formulation and delivery method could be addressed to increase in vivo treatment efficacy. Taken together, these data support that Lactea Lf is a potentially new candidate for further studies as a broad-spectrum antimicrobial medical countermeasure with efficacy against several high priority biodefense-related bacterial pathogens.

Keywords: Burkholderia; Francisella; biofilm; glanders; lactoferrin; melioidosis; tularemia.

Figure 1

Biofilm formation of Burkholderia is inhibited in the presence of Lactea Lf. The BSL-2 surrogates (A) B. thailandensis and (B) B. pseudomallei JW270 or (C) virulent B. pseudomallei ATS2021 were seeded at 10⁵ CFU/well and cultured statically in the presence of Lactea Lf at the indicated concentrations. After 24h, planktonic bacteria were removed, and biofilm formation was quantified by crystal violet staining (left axis). Additionally, percent growth of each condition was calculated, using OD₆₀₀, with respect to the growth of 0 μg/ml (right axis). Error bars represent the standard error of the mean from at least three independent experiments. **P<0.01; ***P<0.001.

Figure 2

Lactea Lf impacts planktonic in vitro growth of Burkholderia in a dose-dependent manner. Dose response curves to Lactea Lf were generated for BSL-2 surrogates (A) B. thailandensis and (B) B. pseudomallei JW270 (seeded at 10⁵ CFU/well) as well as (C) virulent B. pseudomallei ATS2021 and (D) B. mallei ATCC 23344 (seeded at 10⁶ CFU/well) grown with shaking over the course of 40 h Growth was measured by OD₆₀₀ for at least three technical replicates in each experiment. Error bars represent the standard error of the mean from three independent experiments. *P<0.05; **P<0.01; ***P<0.001.

Figure 3

F. tularensis growth is inhibited in a dose-dependent manner in the presence of Lactea Lf in nutrient replete medium. Dose response curves to Lactea Lf were generated for (A) BSL-2 surrogates F. novicida and (B) F. tularensis LVS as well as (C) virulent F. tularensis Schu S4, and (D) F. tularensis OR-96243 (seeded at 10⁷ CFU/well) grown with shaking over the course of 40 h in CDM. Growth was measured by OD₆₀₀ for at least three technical replicates in each experiment. Error bars represent the standard error of the mean from three independent experiments. **P<0.01; ***P<0.001.

Figure 4

F. novicida biofilm formation and F. tularensis growth is inhibited in a dose-dependent manner with increased sensitivity observed in medium that mimics host-adaptation. The BSL-2 surrogate F. novicida (A) was seeded at 10⁷ CFU/well and cultured statically in the presence of Lactea Lf at the indicated concentrations. After 24h, planktonic bacteria were removed, and biofilm formation was quantified by crystal violet staining. Additionally, percent growth of each condition was calculated, using OD₆₀₀, with respect to the growth of 0 μg/ml. Error bars represent the standard error of the mean from at least three independent experiments. Dose response curves to Lactea Lf were generated for (B) BSL-2 surrogate F. novicida and (B) as well as (C) virulent F. tularensis Schu S4, and (D) F. tularensis LVS (seeded at 10⁷ CFU/well) grown with shaking over the course of 40 h in BHI supplemented with 1% IsoVitaleX which is known to promote a host adapted state. Growth was measured by OD₆₀₀ for at least three technical replicates in each experiment. Error bars represent the standard error of the mean from three independent experiments. *P<0.05; **P<0.01; ***P<0.001.

Figure 5

Lactea Lf does not protect mice challenged intranasally with F. tularensis LVS. Groups of mice (n=16) were provided the indicated amount of Lactea Lf intranasally 2 days prior to challenge through 5 days post challenge. Mice were challenged with approximately 1 LD₅₀. (A) Schematic of the study design. Created with BioRender; Biryukov, S. (2025) https://BioRender.com/ras7ige. (B) At day 3 and 6 post challenge, the CFU present in the spleen and lung tissue from three mice determined for each dose. Bars represent the mean value for each treatment. (C) and the remaining 10 mice from each group were monitored for 21 days to record survivorship.

Figure 6

Fluorescently tagged Lactea Lf reaches peak intensity at 1 h post-administration when delivered intranasally. Naïve mice were administered fluorescent tagged Lactea Lf intranasally at the indicated doses and monitored using IVIS for 6 h. (A) A representative mouse administered with 50 mg/mL of Lactea Lf is shown at 1 h post administration. (B) Quantification of signal over time post administration. Data represent the average of three mice. Error bars represent the standard error of the mean.

Figure 7

Lactea Lf localizes in the NALT post-administration of treatment. Tissues were harvested from mice administered fluorescent tagged Lactea Lf intranasally at 6 hours post-treatment and the fluorescent signal was viewed and quantified using an IVIS system. Data points represent the readings from the listed organs from three mice. Bar represents the average from the three data points.