3.2. Intestinal Microbiota Composition and Stool Histamine Concentration
The intestinal microbiota of HIT and control groups was analyzed and compared in terms of phylum, family, genus and species. The two study groups shared a similar profile of phyla (
Figure 1a), with Firmicutes and Bacteroidetes being the most dominant (approximately 90% of the total gut microbiota). Although without statistical significance, the HIT group showed a slightly higher relative abundance of the phylum Proteobacteria (3.52%) in comparison with the control group (1.88%). Similarly, Schink et al. (2018) reported higher levels of Proteobacteria in patients with histamine intolerance symptoms. According to this work, the intestinal overgrowth with Proteobacteria could result in a low-grade intestinal inflammation that could lead to epithelial dysfunction. High proportions of this phylum have also been found in patients with different intestinal disorders, such as Crohn’s disease, ulcerative colitis, colorectal cancer and IBS [
27,
28,
29,
30]. Intestinal inflammation may increase the amount of oxygen available in the intestinal lumen, resulting in a shift from obligate anaerobic bacteria towards facultative anaerobic bacteria, such as Proteobacteria. Consequently, an increase in its abundance has been postulated as a hallmark of dysbiosis [
31,
32].
Regarding bacterial families,
Lachnospiraceae (Firmicutes),
Ruminococcaceae (Firmicutes) and
Bacteroidaceae (Bacteroidetes) represented more than 50% in both the control and HIT groups (
Figure 1b). Statistically significant differences between groups were observed in four bacterial families (
p < 0.05) (
Table 3). For example, a lower abundance of
Prevotellaceae (Bacteroidetes) was found in the HIT group. An under-representation of several members of this family group may indicate reduced mucin synthesis, which has been associated with increased gut permeability [
33]. Schink et al. (2018) reported higher mean values of a marker of intestinal permeability (zonulin) in histamine intolerance patients in comparison with those recommended for the healthy population, suggesting a mild alteration of gut permeability in these patients [
23]. According to these authors, an increased gut permeability facilitates the penetration of microbial metabolites, such as histamine, and, in turn, could lead to histamine-associated symptoms. Additionally, bacteria belonging to the
Prevotellaceae family have been associated with a range of functions in the organism, such as interaction with the immune system and the synthesis of thiamine, folate and short-chain fatty acids [
33].
All the genera and species identified in the microbiome of both study groups are shown in the
Supplementary Material (Supplementary Tables S1 and S2). Overall, statistically significant differences were found in the relative abundance of 21 genera and 30 species between control and HIT groups (
p < 0.05) (
Figure 1c,d). In their study with histamine-intolerant patients, Schink et al. (2018) reported significant differences in five bacterial genera, including only those with an abundance greater than 0.01% [
23]. Applying the same criterion, in the present study, more differences were identified at the genus level (up to nine).
Although the relative abundance of
Ruminococcus in the control group was highly variable, it was statistically significantly lower in individuals with histamine intolerance (
p < 0.05) (
Figure 2).
Ruminococcus is thought to play a role in maintaining a healthy human gut [
34]. Members of this genus can degrade complex polysaccharides into a variety of simple sugars, making them more available for the epithelium cells of the large intestine [
34,
35]. The relative abundance of the genus
Faecalibacterium (
Figure 2), especially the species
Faecalibacterium prausnitzii (
Figure 3), was also significantly lower in the HIT group (
p < 0.05). Proposed as a marker of gut health,
F. prausnitzii is one of the most prevalent and abundant producers of butyrate in the human gut, a short-chain fatty acid that represents the main energy source for colonocytes, and it displays protective properties against colorectal cancer and inflammatory bowel diseases [
36,
37,
38]. Regarding
Bifidobacterium and
Lactobacillus (
Figure 2), two other bacterial genera frequently associated with good intestinal health, no significant differences were found between the two groups. Only two species displayed a lower mean relative abundance in the HIT group (
Bifidobacterium adolescentis,
p = 0.034 and
Lactobacillus rogosae,
p = 0.017) (
Supplementary Table S2).
Conversely, the genera
Staphylococcus and
Proteus were significantly more abundant in the HIT group (
p < 0.05), with mean values 7- and 1.8-fold higher than in the control group, respectively (
Figure 2). Several bacteria from these genera have shown an important capacity to form histamine [
39,
40,
41]. Moreover, members of the family
Enterobacteriaceae, known to be among the most prolific histamine-producing bacteria, were also significantly more abundant in the HIT group (
Figure 2) [
39,
40,
41], although they could not be identified at the genus level. It should be mentioned that the ability to form histamine is reported to be strain-dependent [
42].
To date, studies on histamine-producing bacteria have been mainly focused on strains isolated from food samples. However, the histaminogenic capacity of the gut microbiota has been studied only recently, and data are still limited [
32,
43,
44]. A systematic in silico search published in 2021 identified 117 species with a putative histamine-secreting capacity within the human gut microbiome [
32], many of them belonging to genera extensively reported as histaminogenic, such as
Morganella,
Lactobacillus,
Staphylococcus,
Photobacterium and
Clostridium [
24,
41,
42]. For example, according to Mou et al. (2021),
Clostridium perfringens is one of the species most frequently associated with the enzyme histidine decarboxylase, regardless of strain [
32]. In the present study, the occurrence of
C.
perfringens (
Figure 3), a bacterium responsible for several gastrointestinal disorders, was more frequently identified in the HIT group and only in two healthy individuals. Similarly, the abundance of
Enterococcus faecalis,
Proteus mirabilis and
Escherichia coli tended to be higher in the HIT group (
Figure 3). These species were isolated from the human gut by Pugin et al. (2017) and identified as producers of histamine as well as other biogenic amines, such as putrescine, cadaverine and tyramine [
24].
It has been suggested that histamine secreted by the gut microbiota could have an impact on the health or disease status of the host. In the present study, the higher abundance of intestinal histaminogenic bacteria found in histamine-intolerant patients could have resulted in an excess accumulation and systemic absorption of histamine. Notably, patients with histamine intolerance frequently suffer from DAO deficiency, which could also enhance the toxicity of intestinal histamine. Additionally, an excess of histamine could negatively affect the inflammatory state of the intestinal mucosa. High amounts of histamine secreted by gut bacteria were linked to a proinflammatory response and signs of deteriorating health in murine specimens [
43,
45,
46]. Mishima et al. (2020) suggested that intestinal dysbiosis, involving an over-representation of histamine-secreting bacteria and higher intestinal histamine levels, was potentially associated with the development and aggravation of IBS [
47]. After analyzing 2451 stool metagenomes, Mou et al. (2021) also found that putative histamine-secreting bacteria were significantly enriched in patients with ulcerative colitis and Crohn’s disease [
32]. According to these authors, the enrichment of histamine-secreting species in IBD patients was not attributed to a single taxon but highly dependent to the cohort characteristics. The involved bacterial taxons included Actinobacteriota, Firmicutes, Proteobacteria and Bacteroidiota, depending on the study; some of them were also found to be increased in the HIT patients from the current study [
32]. Another study observed a higher abundance of histamine-producing bacteria in adults diagnosed with asthma [
48]. These previous studies, together with the current results, support the potential association between histamine-secreting bacteria and the inflammatory status occurring in this kind of disorder. In contrast, some studies, both in vitro and in murine models, have demonstrated that intestinal histamine exerts immunomodulatory effects by suppressing the production of proinflammatory interleukines [
49,
50].
Concerning the histamine concentration in stools, no significant differences were found between study groups (
p = 0.681). As shown in
Figure 4, the majority of both healthy and histamine-intolerant individuals (71% and 92%, respectively) displayed fecal histamine levels within the normal range (<959 ng/g stool). The obtained results are in accordance with those of Schink et al. (2018), who also found very similar histamine levels among stool samples [
23]. Therefore, the increased presence of histamine-secreting bacteria found in the HIT group was not associated with a higher histamine excretion in feces.
3.3. Bacterial Diversity
Bacterial species diversity was evaluated through indices of alpha diversity (Shannon and Simpson indices) and beta diversity (multidimensional scaling by PCoA and Bray–Curtis dissimilarity). Regarding alpha diversity, which is a measurement of the mean species diversity within the human gut, no significant differences were observed between the HIT and control groups for any of the evaluated indices (Shannon index,
p = 0.411 and Simpson index,
p = 0.681).
Figure 5 shows the number of identified species belonging to the main genera that differed significantly in abundance between the two groups. Although the HIT group showed a significantly different proportion of genera with the capacity to form histamine (
Staphylococcus and
Proteus) and genera considered as a biomarker of a healthy gut (
Ruminococcus and
Faecalibacterium), these differences were not observed in terms of species number. However, a lower diversity in
Bifidobacterium and
Lactobacillus species was observed in the HIT group, with individuals showing only 69% and 59% of the species found in the control group, respectively (
Figure 5).
In disagreement with our results, Schink et al. (2018) found a lower alpha diversity in a group of 8 histamine-intolerant individuals in comparison with 10 healthy subjects [
23]. Similar discrepancies exist in studies on other types of food intolerance or gastrointestinal disorders, some observing a reduced alpha diversity in patient groups [
51,
52,
53] and others reporting no differences in this parameter [
54,
55].
Beta diversity refers to the interindividual differences in the distribution pattern of genera and species. In this case, beta diversity determined by the Bray–Curtis index showed statistically significant differences between the two groups, both for genera (
p = 0.024) and species (
p = 0.029). As shown in
Figure 6, the samples of the HIT group are more scattered compared to those of healthy individuals, which denotes a higher degree of heterogeneity in their intestinal microbiota.