An adaptable and non-invasive method for tracking Bifidobacterium animalis subspecies lactis 420 in the mouse gut

https://doi.org/10.1016/j.mimet.2021.106302Get rights and content

Highlights

  • Bifidobacterium or Lactobacillus genera improve outcomes in diseased heart models.

  • Indocyanine green (bio)fluorescently labeled Bifidobacterium 420 in vitro and in vivo.

  • Contrary to previous thoughts, the probiotic did not colonize any region of the gut.

Abstract

Probiotic strains from the Bifidobacterium or Lactobacillus genera improve health outcomes in models of metabolic and cardiovascular disease. Yet, underlying mechanisms governing these improved health outcomes are rooted in the interaction of gut microbiota, intestinal interface, and probiotic strain. Central to defining the underlying mechanisms governing these improved health outcomes is the development of adaptable and non-invasive tools to study probiotic localization and colonization within the host gut microbiome. The objective of this study was to test labeling and tracking efficacy of Bifidobacterium animalis subspecies lactis 420 (B420) using a common clinical imaging agent, indocyanine green (ICG). ICG was an effective in situ labeling agent visualized in either intact mouse or excised gastrointestinal (GI) tract at different time intervals. Quantitative PCR was used to validate ICG visualization of B420, which also demonstrated that B420 transit time matched normal murine GI motility (~8 hours). Contrary to previous thoughts, B420 did not colonize any region of the GI tract whether following a single bolus or daily administration for up to 10 days. We conclude that ICG may provide a useful tool to visualize and track probiotic species such as B420 without implementing complex molecular and genetic tools. Proof-of-concept studies indicate that B420 did not colonize and establish residency align the murine GI tract.

Graphical abstract

Workflow diagram of ICG-B420 incubation to murine administration followed by ICG fluorescence visualization and qPCR quantitation.

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Introduction

The gut microbiome is a large and diverse community of microorganisms that inhabit and interact with the human intestinal lining (Putaala et al., 2010). The unique biological relationship between gut microbiota and its host is termed symbiosis under healthy conditions. This symbiotic relationship is preserved by a mucosal layer containing both antimicrobial peptides and innate lymphoid cells. Disruption of symbiosis, or dysbiosis, can instigate detrimental interactions between colonic microbiota, their metabolites, and the host immune system activating the innate immune system and instigating a pro-inflammatory response (Brown et al., 2013). Dysbiosis of the microbiome is associated with several metabolic disorders including obesity, diabetes, and cardiovascular disease (Karlsson et al., 2013). Many studies demonstrate that host-microbiota symbiosis is maintained by microbiota and microbiota-derived metabolites in gut, which lay in close proximity to immune-directing tissue (Bauer, 2018a, Bauer et al., 2018b, Zadeh-Tahmasebi et al., 2016). These microbiota-derived factors impact metabolic processes including nutrient sensing and whole-body glucose regulation. For instance, the gut shows high expression of bacterial-derived transcripts for carbohydrate metabolism, suggesting a central role in this process (Zoetendal et al., 2012).

The mechanism underlying the impact of dysbiosis on disease is not well understood in part due to the complexity of the gut microbiota and the gut environment (Mowat and Agace, 2014, Tremaroli and Bäckhed, 2012). In humans, the gastrointestinal (GI) tract stretches over 20 m with an intestinal surface area 100 times greater than body surface area (DeSesso and Jacobson, 2001). Additionally, the GI tract is home to several unique habitats. Due to changing physiological needs, portions of the small and large intestines differ significantly in pH, bile acid concentration, and oxygen content (Donaldson et al., 2015). These conditions, along with available fuel sources, determine the amount and type of bacteria that can survive in a given section of the GI tract (Derrien et al., 2010, Stearns et al., 2011). Given this, certain probiotic strains may be preferentially equipped to survive and act in certain sections of the GI tract.

Probiotics are defined by the World Health Organization as “live microorganisms that confer a health benefit to the host when consumed in adequate amounts (Sanders, 2011).” A common probiotic strain is Bifidobacterium, a highly diverse gram-positive bacteria from the phylum Actinobacteria and Bifidobacterium genus found in oral cavities, GI tracts and dairy products (Fang and Gough, 2013, Lee and O’Sullivan, 2010, Milani et al., 2013, Wosse et al., 1977). Previous work demonstrates that Bifidobacterium animalis subspecies lactis 420 (B420) positively impacts metabolic syndrome by limiting weight gain, improving glucose metabolism, and reducing low-grade inflammation (Hotamisligil, 2017, Putaala et al., 2008, Stenman et al., 2014a, Stenman et al., 2016). In our studies, pre-administration of B420 following a myocardial infarction (MI), a known inflammatory disease, attenuated cardiac damage (Danilo et al., 2017). To better elucidate the mechanisms of probiotics, specifically B420, we must consider not only how but also where probiotics elicit their actions. Probiotic colonization, localization, and residence in the GI tract have important implications for determining mechanism of host-microbe interaction. Understanding these basic mechanisms will impact characteristics such as dose, frequency, and length of probiotic administration, critical factors when utilizing B420 and other probiotic strains for therapeutic use.

Probiotic transit, adherence and colonization in both small and large intestines are understudied areas; previous research largely focused on detecting probiotic species in feces and large intestine. Visualization through fluorescence is challenging or not possible due to poor genetic accessibility and lack of vector systems for Bifidobacterial genomes (Dominguez and O’Sullivan, 2013, Grimm et al., 2014, Lee and O’Sullivan, 2010, Wiles et al., 2006). Therefore, the central goal of this proof-of-concept study was to develop an efficient, cost-effective technique to visualize and track probiotic transit through the GI tract in as little as a single digestive cycle in a mouse.

We hypothesized that labeling of microbial species with an externally detectable marker would allow detection as to their specific geographic residence/location during transit through the gut. First, we incubated B420 with common, FDA-approved contrast agents, ISOVUE-300 or indocyanine green (ICG), and visualized by x-ray fluoroscopy (ISOVUE-300) or fluorescence (ICG) along the GI tract at different timepoints. Further, we validated and quantified B420 using quantitative PCR at different regions of the GI tract. We found that ICG was a more effective labeling agent over ISOVUE-300, and following a single bolus of B420, we also demonstrated B420 transit time matched normal murine gut motility of approximately 8 h. Importantly, we demonstrated that B420 did not colonize the host gut microbiome, even following 10 consecutive days of B420 administration, where the amount of detectable B420 diminished to control levels within 24–48 h.

Section snippets

Incubation of B420 with ICG

A determination of B420 stability and B420 growth curve was executed using purified stock of B420 (see Supplemental Data, Fig. S1). A serial dilution of 1 mg/ml solution (the solubility concentration of ICG in water) from 1 mg/ml to 0.001 mg/ml, was plated in a 96 well plate and imaged to optimize the ICG fluorescent signal. Based on this, 0.01 mg/ml was used to label 12-h cultured bacteria for 1 h in dark conditions. The pellets were then centrifuged at 16,000 xg and washed three times in

Visualization of ICG-labeled B420

B420 incubated with ISOVUE was detectable by x-ray fluoroscopy ex vivo. However, the ISOVUE-300 signal was not sensitive enough to track B420 in vivo (see Supplemental data, Fig. S2). Therefore, we moved to another FDA-approved agent, ICG, a contrast agent regularly used to visualize arteries and microvasculature in the brain and skin (Alander et al., 2012). In the following series of in vitro experiments, we wished to determine the toxicity of ICG to B420 growth and proliferation while

Discussion

Advancing potential therapeutic uses of exogenously delivered probiotic species may depend on the ability to measure the rate, location, and abundance of these species as they transit through the gut. Key findings from our study include: (1) ICG was a more effective labeling agent over ISOVUE-300 for the bacterial strain tested, B420; (2) B420 transit time matched murine gut motility, and (3) B420 did not adhere and colonize the intestinal mucosa of the host gut microbiome. Following ingestion,

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Institutes of Health (NIH) grant HL098256, the American Heart Association (AHA) grant 16GRNT31390006, and independent and interdisciplinary trainings in biomedical engineering and cardiovascular sciences (5T32HL00795515, 5T32 HL007249, and T32 GM132008). Support was also received from the University of Arizona Sarver Heart Center.

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