Background
Gut microbiota has been closely linked with many chronic and refractory diseases such as inflammatory bowel disease, colorectal cancer, obesity, diabetes, and even mental diseases like autism. The key roles of an increasing number of gut bacteria have been revealed, which herald a great progress in our knowledge of the etiology of those diseases and offer great promise for optimizing health and treating diseases in novel ways. For instance, an
Enterobacter cloacae strain has been identified as an obesity-inducing opportunistic pathogen, whose mono-association in germfree mice can recapitulate the obese phenotype, including low-grade inflammation, adiposity and insulin resistance [
1]. Another bacterium,
Akkermansia muciniphila has been demonstrated to negatively correlated with symptoms of obesity and type 2 diabetes, and has the potential as a probiotic to reverse high-fat diet-induced metabolic disorders in mice [
2]. Recent advances in microbial cultivation and model animal are greatly facilitating our understanding of the fundamental and ultimate question—”who” does “what” and “how” referring to the active roles and mechanisms of human gut microbiota [
3].
Bilophila wadsworthia is a sulfite-reducing and hydrogen sulfide-producing microbe, which was originally isolated from specimens of peritoneal fluid and tissue of patients with appendicitis [
4], while usually difficult to detect in healthy individuals.
B. wadsworthia is an obligately anaerobic Gram-negative bacillus and can be stimulated by bile. Since it was named in 1989, there has been limited progress on this bacterium. Till in 2012, researchers began to realize the function of this commensal gut bacterium. Devkota reported that a milk-derived saturated fat diet induced a bloom of
B. wadsworthia in gut of SPF mice. And mono-inoculation of this pathobiont in germfree IL10
−/− mice fed with milk fat diet can even induce T
H1 immune response and colitis development [
5]. This bacterium was also detected over-represented in colonic microbiota of colorectal cancer patients, which implied its possible role in colorectal carcinogenesis [
6]. Though mounting studies have highlighted the correlations of
B. wadsworthia in different human diseases [
7‐
9], especially chronic metabolic diseases, the mechanisms of its pathogenicity are not yet well characterized.
In the study described here, we obtained an isolate of B. wadsworthia from a new-onset LADA (latent autoimmune diabetes in adults) patient. The objective of this study was to examine the outcome of the infection of this strain in normal SPF mice, and try to elicit its possible pathogenicity.
Discussion
Bilophila wadsworthia belongs to the
Desulfovibrionaceae family and is the second most recovered isolate of sulfidogenic bacteria in human gut. Sulfidogenic bacteria metabolize sulfated compounds and produce hydrogen sulfide that triggers direct inflammation, exerts genotoxic and cytotoxic effects on epithelial cells, and impairs gut barrier [
22]. Correlation of sulfidogenic bacteria to the etiology of chronic metabolic diseases such as obesity [
23], type 2 diabetes [
24] and colorectal cancer [
6] has been explored only recently.
Though
B. wadsworthia have been identified from a variety of intra-abdominal infections, including sepsis, liver abscesses, cholecystitis et al. [
25,
26], the direct evidence linking
B. wadsworthia with chronic diseases is still insufficient. In the present study described here, we investigated the outcome of
B. wadsworthia infection on SPF C57BL/6 mice. We hypothesize that increased numbers of
B. wadsworthia in gut microbiota induces systemic inflammation and thus contributes to the onset of the chronic diseases.
After inoculation, we found that the
B. wadsworthia strain couldn’t be detected from fresh fecal samples of mice with only one time oral gavage. This refers to a typical microbial ecology phenomenon termed colonization resistance (CR), which is a mechanism that the robust commensal microbiota of the host protects itself against incursion by exogenous and often pathogenic microorganisms [
27]. Only in animals whose CR was impaired by antibiotic treatments or in germ-free animals without any microbiota, the hosts showed susceptible to invading pathogens even at a very low dosage [
28]. To solve this problem, we repeated exposure of
B. wadsworthia to mice for 1 week to ensure enough number of live
B. wadsworthia cells in the gut. We observed that this strain could survive through the digestive tract as live
B. wadsworthia could be recovered from fresh feces of mice. Such a strategy has been used in many studies to increase the adaptability of the inoculated bacterium to experimental SPF mice [
2,
19].
It has been proved that milk-derived or lard-based high saturated fat diet [
5,
29] and even an animal-based diet [
30] can markedly promote the flourish of
B. wadsworthia in gut. Saturated fat diet leads to increased hepatic taurine conjugation of bile acids, thus provides more sulfur-rich taurocholic acid in gut, which accelerates the growth of the sulfite-reducing bacterium
B. wadsworthia. In this study, all the mice were fed with normal chow diet, that didn’t favor the growth of
B. wadsworthia in gut. This may also contribute to the difficulty to adapt this strain to gut of mice.
In the present study, we observed that
B. wadsworthia could induce pathological responses to the SPF mice. Seven days of inoculation resulted in a significant decrease of body weight and three main fat mass of BW mice, compared with NC mice. While, the weight of spleen and liver of BW mice showed a reverse trend that animals in this group developed demonstrable hepatosplenomegaly, which is a common clinico-pathological sign related with various infections [
31‐
33].
We also examined the response of colon after
B. wadsworthia inoculation. All animals had normal colonic morphology and no obvious histologic changes were observed. McOrist et al. also reported that
B. wadsworthia infected pigs displayed no or minor specific lesions in the small and large intestine [
34]. The relative gene expression of pro-inflammatory cytokines IL-6, TNF-α and TLR4 in colon of BW mice displayed similar levels with those of NC mice, suggesting no local immunopathological response induced in colon by
B. wadsworthia. A previous work has presented that consumption of milk-derived fat not only promoted the expansion of
B. wadsworthia in SPF mice, but also increased incidence of colitis in colon of genetically susceptible
Il10
−/− mice, while not in wild-type mice [
5]. Our result also demonstrated that the
B. wadsworthia infection in normal SPF mice cannot directly trigger immune response in colon in the present short-term experiment.
Notably, we observed that
B. wadsworthia infection provoked a systemic inflammatory response in SPF mice. The key circulating inflammatory cytokines like SAA and IL-6 significantly increased after
B. wadsworthia inoculation. SAA proteins are acute phase proteins mainly secreted by the liver in response to pro-inflammatory stimuli [
35], and their increased levels in serum have been associated with several chronic inflammation-based diseases, such as obesity [
36,
37], hyperglycemia [
38], insulin resistance [
39] and cardiovascular disease [
40,
41]. SAA has also been reported to be involved in LPS signaling pathway that links inflammation to metabolic disorders in mice, for instance, de Oliveira et al. found that high fat diet induced metabolic endotoxaemia, and, body weight gain and insulin resistance could be prevented by an SAA-targeted antisense oligonucleotide treatment [
42]. IL-6 also represents a keystone cytokine in infection and inflammation [
43], which elicits cellular immune responses to affected cells and mucosal humoral responses directed against reinfection. Increasing serum levels of IL-6 provides the basis for the amplification step of chronic inflammatory proliferation [
44]. We also detected the levels of serum LBP and TNF-α, their levels in BW group tended to be higher than NC group, while the differences were not statistically significant. Similar phenomenon has also been reported in both humans and rats, that the levels of IL-6 and TNF-α were in parallel with the abundance change of
Bilophila wadsworthia [
8,
45].
As a Gram-negative bacterium,
B. wadsworthia can release lipopolysaccharide as endotoxin, though its endotoxic activity is relatively low compared with
E. coli [
46]. This strain can also induce procoagulant, and an in vitro study implied its ability to attach to human epithelial cells of the colon, that is usually considered as the first step in establishing infection in the host [
46,
47]. Besides,
B. wadsworthia participates in taurine respiration in human gut which leads to sulfite formation [
48]. The metabolic end-product of dissimilatory sulphate and sulfite reduction, hydrogen sulfide, is well documented to be pro-inflammatory and toxic to mucosal tissue at higher physiological doses [
49]. These features of
B. wadsworthia may all contribute to its inflammatory responses and pathogenicity.
Recent years, accumulating evidence points toward the interplay between the microbiota and the immune system as central to the development of chronic metabolic diseases [
50‐
52]. The feature that the outgrowth of
B. wadsworthia in gut microbiota can promote low-grade systemic inflammation may account a part for the high prevalence of this bacterium in stool of mice with chronic disease such as colon cancer, colitis and low-fat/high-sugar diet induced obese reported previously [
5,
6,
8]. Though the
B. wadsworthia strain used in this study was isolated only from one LADA patient, and its prevalence in other patients is unclear, the data showed here provided important clue for the physiology of this species of commensal bacteria. More patients will be recruited in future study to further confirm the results.
To investigate whether the etiology of
B. wadsworthia is related to its perturbation of the host gut microbiota, we compared the composition of gut microbiota in mice with and without
B. wadsworthia infection. It was demonstrated that
B. wadsworthia made no significant change to the whole existing gut microbiota, in terms of not only the α-diversity but also the OTUs composition. This result indicates that, after 7 days of
B. wadsworthia infection, the inflammatory phenotypes developed in mice might be caused by
B. wadsworthia itself and/or its metabolic products, without marked modification of the host’s overall microbiota. Similar results have been reported in several other studies. For example,
A. muciniphila is a mucin-degrading bacterium that resides in the human gut [
53], its abundance decreases in obese and type 2 diabetic mice. Four weeks of
A. muciniphila administration could reverse high-fat diet-induced metabolic disorders in C57BL/6 mice, without modifying gut microbiota composition [
2]. A butyrate-producing bacterium,
Anaerostipes hadrus BPB5, could increase the butyrate content in healthy SPF C57BL/6 mice after 7 days treatment, while the whole gut microbiota didn’t change significantly [
19].
Our results add new evidence to the virulence of B. wadsworthia. The decreased body weight and fat mass, apparent hepatosplenomegaly and elevated serum levels of SAA and IL-6 demonstrated that this bacterium triggers the systemic low-grade inflammation of the host. Unfortunately, there is no diabetes mellitus specific parameters addressed in this report. Our next plan is to examine the clinical outcomes of B. wadsworthia including glucose and insulin metabolism, to comprehensively understand the metabolic activities and functions of it.