jOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2018, 69, 4, 579-593

[Link] | DOI: 10.26402/jpp.2018.4.09

M. SCHINK1, P.C. KONTUREK2, E. TIETZ1, W. DIETERICH1, T.C. PINZER3, S. WIRTZ3, M.F. NEURATH3, Y. ZOPF1

MICROBIAL PATTERNS IN PATIENTS WITH HISTAMINE INTOLERANCE

1
First Department of Medicine, Hector Center for Nutrition, Exercise and Sports, Friedrich-Alexander-Universitaet Erlangen-
Nuernberg, Erlangen, Germany; 2Second Department of Medicine, Thuringia-Clinic Saalfeld, Saalfeld/Saale, Germany; 3First
Department of Medicine, Friedrich-Alexander-Universitaet Erlangen-Nuernberg, Erlangen, Germany

Histamine intolerance represents a controversially discussed disorder. Besides an impaired degradation of orally supplied
histamine due to diamine oxidase (DAO) deficiency, a deranged gut flora may also contribute to elevated histamine levels.
Our aim was to determine the intestinal bacterial composition in patients with proven histamine intolerance in comparison
to other food intolerances and healthy controls. A total of 64 participants were included in the study, encompassing 8 patients
with histamine intolerance (HIT), 25 with food hypersensitivity (FH), 21 with food allergy and 10 healthy controls (HC). All
participants underwent blood testing for total and food-specific immunoglobulin E, plasma histamine and DAO serum
activity. Stool samples were used to analyze stool histamine and zonulin levels and bacterial composition by 16s rRNA
sequencing. No significant differences in stool histamine levels were observed, but HIT patients showed elevated levels of
stool zonulin. Microbiota analysis revealed increased levels of Proteobacteria (5.4%) and a significantly reduced alpha-
diversity in the HIT group (P = 0.019). On family level, HC showed a significantly higher abundance of Bifidobacteriaceae
compared to other study groups (P = 0.005), with lowest levels in the HIT group (P = 0.036). Also significantly reduced
abundances of the genera Butyricimonas (P = 0.026) and Hespellia (P = 0.025) were observed in the HIT patients, whereas
Roseburia were significantly elevated (P = 0.021). We concluded that the altered occurrence of Proteobacteria and
Bifidobacteriaceae, reduced alpha-diversity as well as elevated stool zonulin levels suggest a dysbiosis and intestinal barrier
dysfunction in histamine intolerant patients, which in turn may play an important role in driving disease pathogenesis.

K e y w o r d s : dysbiosis, food intolerance, gastrointestinal microbiome, histamine, intestinal barrier, diamine oxidase, lactic
acid bacteria

INTRODUCTION histamine concentrations and evoking the described symptoms

by affecting various organ systems (e.g. cardiovascular system,
The prevalence of patients suffering from gastrointestinal respiratory tract, skin, nervous system, intestine) (4, 5).
and extra-intestinal afflictions after food ingestion is rising. The However, also other factors are discussed to affect histamine
spectrum of food intolerances reaches from carbohydrate intolerance, for example an alteration of the intestinal bacteria.
malabsorption (e.g. lactose, fructose) to immunological IgE or Various bacteria, which are able to convert histidine from
non-IgE-mediated food allergies (1, 2). In addition, histamine proteins into histamine, naturally occur in the digestive tract as
intolerance (HIT) is also often considered to be responsible for part of the normal intestinal gut flora (6, 7).
gastrointestinal symptoms after food intake. Thereby, histamine Interestingly, some probiotic strains including several lactic
intolerance is defined as an adverse reaction of ingested acid bacteria, like Lactobacillus reuteri, Lactobacillus casei and
histamine that affects different organ systems and results in Lactobacillus delbrueckii subsp. bulgaricus, possess the enzyme
various intestinal and extra-intestinal symptoms (3). Ingestion of histidine decarboxylase (HDC) and are therefore able to generate
histamine containing foods and beverages, including fish, cheese biogenic amine (8, 9). The presence of these bacteria in the
or red wine, are supposed to trigger symptoms like flush, human intestine might contribute to increased histamine levels
pruritus, nausea, vomiting, diarrhea and abdominal pain (3). and promote histamine sensitivity in some persons.
Other foods like citrus fruits or various drugs further contribute It is well known that alterations of the human intestinal
to an elevated histamine concentration through their histamine- microbiota are linked to various diseases. Besides obesity or
liberating effect (4). Although the exact mechanism of the cardiovascular disease (10, 11), a dysbiosis is discussed in the
pathogenesis is still unclear, a reduced intestinal diamine oxidase pathogenesis of different autoimmune diseases including type 1
(DAO) activity, which is important for degradation of diabetes, rheumatoid disease, inflammatory bowel disease or
exogenously supplied histamine, is presumed (4). This leads to celiac disease (12, 13). But even in patients with an allergy, the
an insufficient degradation of food derived histamine, which influence of the microbiota as a triggering factor for asthma and
passes into the blood stream leading to increased plasma food allergy is discussed (14, 15). Several studies revealed a
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correlation between a low microbial exposure in childhood and without IgE antibodies and without validated histamine
an increased risk for allergies. Thereby, multiple factors can intolerance, but clinical symptoms including abdominal pain,
influence this risk positively or negatively by altering the diarrhea, nausea, headache, skin changes or allergic rhinitis,
intestinal microbiota, e.g. mode of birth, duration of were classified as food hypersensitive patients (FH group). The
breastfeeding, treatment with antibiotics, infections, living with healthy controls (HC group) showed no clinical symptoms and
older siblings or furred pets (16). Interestingly, the use of no signs for food allergy and histamine intolerance.
probiotics seems to have immunomodulatory effects in allergic The whole diagnostic procedure for group allocation was
disease by suppressing histamine signaling (17). Via the published by Pinzer et al. (19).
induction of regulatory T cells (Tregs) some Clostridia species
seem to suppress symptoms in murine models of intestinal Nutritional assessment
allergy (18). All these facts underline the importance of intestinal
bacteria in human immunity and health. To determine daily intake of macronutrients the Freiburger
To determine the influence of the human gut microbiota in Diet Protocol (Nutri-Science GmbH, Freiburg, Germany) was
the pathogenesis of histamine intolerance, we analyzed the used. Therefore, all study participants were asked to fill in a
intestinal bacterial composition by sequencing the bacterial 16S three-day nutritional dairy at the beginning of the study. Daily
rRNA of stool samples derived from patients with a confirmed intake of energy, carbohydrates, fats, fibers and alcohol was
diagnosis of histamine intolerance. These data were compared evaluated by PRODI® (version 6.5 expert, Nutri-Science
with the microbial patterns of stool samples from healthy GmbH).
individuals, patients with food allergy or food hypersensitivity.
The measurement of histamine concentrations in stool samples Sample collection and analysis
was done to evaluate the histamine production by intestinal
bacteria. To assess the gut permeability, zonulin, a regulator of Venous blood samples were taken from every study
tight junctions, was measured in serum and stool samples. participant and plasma histamine, DAO activity, total IgE
concentration and food-specific IgE antibodies were quantified
in blood serum. Serum zonulin and TNF-α concentrations were
MATERIALS AND METHODS measured by ELISA (IDK® Zonulin ELISA Kit and IDK®
TNF-α ELISA, Immunodiagnostic AG, Bensheim, Germany)
Study participants
following manufacturer’s instructions. Histamine and zonulin
from stool samples were determined with the Histamine ELISA
Patients with histamine intolerance, food hypersensitivity and IDK® Zonulin ELISA from Immunodiagnostic AG,
and allergies were recruited over a 12-month period via the Bensheim, Germany. Stool samples were collected once at study
outpatient clinic for nutritional medicine of the Medical beginning from all study participants and were immediately
Department 1 of the University Hospital Erlangen as well as cooled at 4ºC and stored within 4 hours at –20ºC till analysis.
social media platforms. Healthy controls were recruited from the Bacterial DNA was isolated with the QIAamp Fast DNA Stool
circle of friends or colleagues. A total of 64 patients were Mini Kit (Qiagen GmbH, Hilden, Germany) following
included in the study. Exclusion criteria were pregnancy, manufacturer’s instructions. DNA concentration was measured
lactation, being underage and current intake of antibiotics, anti- by NanoDrop 2000 (Thermo Fisher Scientific, Wilmington,
histamines or anti-inflammatory medication. USA).
All participants were informed in detail by a doctor about the For 16S-based microbiome analysis, the amplification of the
aim and procedure of the study and gave their written informed V3-V4 region of bacterial 16S rRNA was realized by NEBNext
consent prior study inclusion. The study was approved by the Q5 Hot Start Hifi PCR Master Mix (New England Biolabs,
ethics committee of the Friedrich-Alexander-University Ipswich, USA). Amplicons were purified with AMPure XP
Erlangen (application number: 231_14B) and in accordance with Beads (Beckman Coulter Genomics, Indianapolis, USA), and
the declaration of Helsinki. the DNA content was measured by fluorometric quantitation
using the Qubit® dsDNA-Kit (Thermo Fisher Scientific,
Group allocation Germany). DNA samples were pooled and analyzed by 2 × 300
bp paired-end sequencing on the Illumina MiSeq platform.
Blood samples were taken of all study participants to Quality control, OTU table generation and taxonomic
determine total immunoglobulin E (IgE) as well as ten food- classification against the database of the ‘Ribosomal database
specific IgEs (chicken’s egg white, milk protein, wheat flour, rye projects’ (RDP, version 16) was performed using Usearch 10 (64
flour, nut mixture, soy bean, tomatoes, salmon, casein and bit version).
celeriac). Participants with gastrointestinal (diarrhea, nausea,
vomiting, abdominal pain) and extra-intestinal symptoms Statistical analysis
(allergic rhinitis, oral allergy syndrome, headache, fatigue, skin
changes, asthmatic symptoms) briefly after food ingestion and Statistical analyses were performed using SPSS version 21
positive serological food-specific IgE antibodies and (IBM SPSS Statistics, Ehningen, Germany) and GraphPad Prism
significantly elevated total IgE (361.2 kUA/L; P > 0.001) were 7 (GraphPad Software Inc., La jolla, CA, USA). Bioinformatics
classified as food allergy patients (FA group). Individuals with analysis for bacterial composition were performed with
symptoms, but negative food-specific IgE antibodies and low METAGENassist (20) and MicrobiomeAnalyst (21).
total IgE levels (< 100.0 kUA/L) underwent further Characteristic data are described as means ± standard deviation
measurements of plasma histamine levels and serum DAO (SD), median and range (min-max) or in number (n) and percent
activity. Patients with impaired histamine degradation, (%). All data for bacterial proportions are described as median
characterized by elevated plasma histamine levels and decreased with minimum to maximum values (min-max). For statistical
DAO activity in serum and an alleviation of symptoms during a evaluation data were checked by Kolmogorov-Smirnov-test for
histamine-free diet, were further validated by repeated blood normal distribution. Differences between study groups were
samples over a period of 24 hours, and allocated to the histamine determined using Kruskal-Wallis-test for non-parametric data.
intolerant group (HIT group). The remaining participants Due to the exploratory character of this pilot study, no correction
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for multiple testing was applied. For categorical variables female) and 10 healthy volunteers (age 31.3 ± 13.9 years, 90%
differences between study groups were analyzed by Pearson’s chi- female) without gastrointestinal complaints were acquired.
squared test. The alpha-diversity was described using Shannon- Patients with proven food allergy showed positive IgE
Weaver-Index (SWI) and Simpson’s reciprocal index (SI). The antibodies against nut mixture (76.2%), wheat flour (47.6%),
Permutational Multivariate Analysis of Variance (PERMANOVA) celery (42.9%), tomato (23.8%), rye flour (23.8%), soybean
was used for analysis of pairwise inter-sample distances with (14.3%), and milk protein (4.8%) as well as significantly
Bray-Curtis method. Correlation analysis of variables was increased total IgE levels (361.2 ± 911.2 kUA/l; P < 0.001).
computed using the non-parametric Spearman rank correlation. None of the individuals from the other groups showed specific
All tests for significance were two sided, and a P-value of IgE antibodies against foods.
P < 0.05 was considered as statistically significant. The presence of further comorbidities including asthma,
atopic eczema, cardiovascular disease, depression, endometriosis,
fibromyalgia, gastrointestinal disease, hypothyreodism was not
RESULTS significantly different between all study groups.
Patients characteristic are shown in Table 1.
Characteristics
Serum and stool parameters
Overall 64 study participants (38.3 ± 14.2 years, 84.4%
female) were enclosed to the study, and classified according to a Patients in the HIT group revealed elevated levels of TNF-α
previous study (19). Briefly, 33 patients (age 38.4 ± 13.4 years, compared to other study groups (P = 0.097) (Table 1). However,
84.8% female) had suspected histamine intolerance. The 24 h only one HIT patient showed a serum TNF-α concentration above
histamine profiling and the measurement of serum DAO activity the reference threshold value of 20 ng/ml (Fig. 1a). Zonulin
revealed 8 out of these 33 patients (12.5%) with histamine levels in stool and serum were similar in all participants (P =
intolerance by definition (decreased DAO activity) and these 0.726 and P = 0.595) (Fig. 1b and 1c), with highest median levels
patients were allocated to HIT-group. The remaining 25 patients for stool zonulin in patients that belong to the HIT or FH groups
(age 41.4 ± 12.8 years; 80.0% female) with normal DAO activity (Table 1, Fig. 1b). Concerning the stool histamine concentrations
were considered as food hypersensitive (FH-group). Additionally, one patient of the FH and one patient of the FA group showed
21 patients with proven food allergy (age 41.4 ± 14.9 years, 81% very high stool histamine levels (> 24.000 ng/ml) (Fig. 1d). The

Fig. 1. Blood and stool parameters of study groups. Fig. 1a shows individual serum TNF-α concentrations with mean values. The
dotted line indicates the reference threshold for normal TNF-α values (< 20 pg/ml). Fig. 1b and 1c show individual zonulin
concentrations in stool (b) and serum (c) with median (horizontal line). The solid lines mark the reference medians and dotted lines
indicate the under and upper threshold of normal concentrations. Fig. 1d shows individual histamine stool concentrations of study
groups with mean values (horizontal line). Dotted line indicates reference threshold for normal histamine values (< 959 ng/ml).
Kruskal-Wallis test was used for multiple comparisons of laboratory values between study groups. Abbreviations: HC, healthy
controls; HIT, histamine intolerants; FH, food hypersensitives; FA, food allergy sufferers.
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Table 1. Patient characteristics of study groups.

Characteristic HC HIT FH FA P-value

Demographic
Amount
10 (15.6) 8 (12.5) 25 (39.1) 21 (32.8) –
[n(%)]
Age
31.3 ± 13.9 28.9 ± 11.2 41.4 ± 12.8 41.4 ± 14.9 0.026*
[years]
Male
1 (10.0) 0 (0.0) 5 (20.0) 4 (19.0)
[n(%)] 0.518
Female
9 (90.0) 8 (100.0) 20 (80.0) 17 (81.0)
[n(%)]
Body mass index
21.2 ± 2.0 24.6 ± 6.5 24.2 ± 4.6 23.4 ± 4.1 0.273
[kg/m²]
Alcohol consumption
8 (80.0) 6 (75.0) 15 (60.0) 17 (81.0) 0.397
[n(%)]
Nicotine abuse
3 (30.0) 1 (12.5) 2 (8.0) 3 (14.3) 0.411
[n(%)]
Probiotic use
0 (0.0) 0 (0.0) 4 (16.0) 1 (4.8) 0.248
[n(%)]

Comorbidities
Asthma
0 (0.0) 0 (0.0) 3 (12.0) 3 (14.3) 0.447
[n(%)]
Atopic eczema
0 (0.0) 0 (0.0) 2 (8.0) 3 (14.3) 0.432
[n(%)]
Cardiovascular disease
1 (10.0) 0 (0.0) 2 (8.0) 1 (4.8) 0.805
[n(%)]
Depression
0 (0.0) 1 (12.5) 2 (8.0) 0 (0.0) 0.357
[n(%)]
Endometriosis
0 (0.0) 1 (12.5) 0 (0.0) 2 (9.5) 0.271
[n(%)]
Fibromyalgia
0 (0.0) 0 (0.0) 2 (8.0) 0 (0.0) 0.359
[n(%)]
Gastrointestinal disease
0 (0.0) 1 (12.5) 3 (12.0) 1 (4.8) 0.578
[n(%)]
Hypothyreodism
0 (0.0) 2 (25.0) 4 (16.0) 6 (28.6) 0.265
[n(%)]
Migraine
0 (0.0) 0 (0.0) 2 (8.0) 0 (0.0) 0.359
[n(%)]

Serum and stool parameters

Total IgE 14.3 ± 9.6 38.7 ± 29.9 25.9 ± 22.7 361.2 ± 911.2 > 0.001
[kUA/l]
TNF-α 3.6 7.4 3.6 5.0 0.097
[pg/ml] (2.7 – 10.3) (3.1 – 22.4) (1.0 – 11.6) (2.3 – 13.2)
Zonulin serum 36.4 31.2 41.2 34.9 0.595
[ng/ml] (12.3 – 48.9) (17.5 – -78.7) (16.6 – 65.8) (9.2 – 161.2)
Zonulin stool 90.0 135.7 85.7 84.6 0.726
[ng/ml] (45.1 – 518.2) (51.2 – 464.2) (38.0 – 305.0) (46.8-198.3)
Histamine stool 283.7 206.0 265.2 196.6 0.828
[ng/ml] (62.6 – 2008.0) (169.8 – 3027.5) (57.9 – 24000.0) (45.7 – 24000.0)

Nutritional intake
Energy intake 2081.7 ± 818.6 2431.3 ± 362.3 2873.3 ± 1218.1 2862.9 ± 1552.8 0.025*
[kcal/d]
Carbohydrates 232.2 ± 104.2 302.4 ± 69.4 291.4 ± 112.1 258.3 ± 94.0 0.097
[g/d (%TE)] (44.5) (50.4) (40.9) (42.1)
Fat 85.9 ± 34.6 86.1 ± 15.9 126.9 ± 66.3 103.5 ± 34.1 0.059
[g/d (%TE)] (37.6) (31.8) (38.4) (37.1)
Protein 79.6 ± 33.0 87.1 ± 24.9 116.1 ± 51.4 94.1 ± 28.2 0.026*
[g/d (%TE)] (15.3) (14.5) (16.6) (16.1)
Fiber 19.4 ± 2.5 31.7 ± 0.0 68.3 ± 81.5 22.8 ± 12.6 0.261
[g/d (%TE)] (1.9) (1.9) (2.2) (1.7)
Alcohol 4.3 ± 2.4 5.0 ± 6.2 13.6 ± 14.7 15.3 ± 9.9 0.028*
[g/d (%TE)] (0.7) (1.0) (2.0) (3.2)
Data are presented as number and proportions (%), mean ± standard deviation. Laboratory values (except total IgE) are expressed as
median and range (min to max). Comparisons between HC, HIT, FH and FA group are assessed by Pearson’s chi-squared test,
respectively, for categorical variables and Kruskal-Wallis test for continuous variables. Statistically significant differences are
indicated by *P < 0.05; ***P < 0.001 and marked in bold type. Abbreviations: HC, healthy controls; HIT, histamine intolerants; FH,
food hypersensitives; FA, food allergy sufferers; %TE, Total energy percent.
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analysis of stool histamine concentrations revealed no significant Microbiome analysis

differences between study groups (P = 0.828) (Table 1).
To compare the microbial composition between different
Nutritional intake study groups, we converted the bacterial counts into percentages.
Bacterial phyla, families and genera with an overall percentage
The nutritional assessment revealed significant differences below 0.01% were excluded from analysis.
in the daily protein (P = 0.026) and alcohol (P = 0.028) intake The microbial patterns showed differences between all
between the study groups (Table 1). Our healthy controls study groups. On phylum level, Bacteroides (61.9%),
ingested lower amounts of protein, reaching significance Firmicutes (31.7%) and Proteobacteria (3.7%) were most
compared to the FH group (P = 0.005). Both, patients of the HIT abundant in all study participants. Significant differences were
group and healthy controls, consumed significantly less alcohol observed for Verrucomicrobia (P = 0.030) with elevated
than patients of the FA group (P = 0.013 and P = 0.023). Daily numbers in patients with FH [0.35% (0.0 – 16.4%)], but minor
carbohydrate intake was highest in the HIT group. proportions in HC [0.02% (0.00 – 2.36%)], HIT [0.00% (0.00
– 0.07%)] and FA groups [0.08% (0.00 – 3.05%)], respectively
Correlation analysis (Fig. 2a, Table 2). Interestingly, patients from the HIT group
showed very low levels of Verrucomicrobia, without any
Correlation analysis between measured blood and stool abundance in five patients reaching significance to FH and FA
parameters via Spearman rank method revealed significant group (P = 0.003 and P = 0.019). In contrast, HIT patients had
correlations. Thereby the stool concentrations of histamine and elevated proportions of Proteobacteria [5.36% (1.34 –
zonulin showed a moderate positive correlation (r = 0.454; 34.59%)] compared to the other study groups, although
P < 0.001). Both, parameters for α-diversity, SWI (r = 0.339; significance was not reached, because of great variations
P = 0.007) and SI (r = 0.337; P = 0.007), were correlated with between the HIT patients (Fig. 2b, Table 2).
TNF-α concentrations.

Fig. 2. Differences on phylum level between study groups. Fig. 2a shows significant higher relative abundance of the phylum
Verrucomicrobia in FH and FA group compared to the HIT group (P = 0.003 and 0.019). Fig. 2b showed highest abundance for the
phylum Proteobacteria in the HIT group [5.36% (1.34 – 34.59%)]. Kruskal-Wallis test was used for multiple comparisons between
study groups. Abbreviations: HC, healthy controls; HIT, histamine intolerants; FH, food hypersensitives; FA, food allergy sufferers.
Significance: *P < 0.05 and **P < 0.01.

Table 2. Phylum level - relative abundance.

Phylum HC HIT FH FA
Bacteroidetes 56.94 (37.94 – 74.03) 63.97 (15.80 – 86.00) 63.94 (27.56 – 83.22) 61.19 (27.56-83.22)
Firmicutes 37.00 (22.29 – 54.78) 25.51 (12.43 – 46.50) 28.87 (14.40 – 58.82) 34.07 (10.63-53.84)
Proteobacteria 3.33 (1.85 – 14.25) 5.36 (1.34 – 34.59) 3.75 (1.22 – 12.26) 4.82 (1.04-11.64)
Actinobacteria 0.45 (0.06 – 3.31) 0.12 (0.01 – 7.39) 0.25 (0.03 – 3.97) 0.13 (0.07-2.86)
Verrucomicrobia 0.02 (0.00 – 2.36) 0.00 (0.00 – 0.07)†,†† 0.35 (0.00 – 16.44)†† 0.08 (0.00-3.05)†
Tenericutes 0.0013 (0.00 – 0.92) 0.00 (0.00 – 0.00) 0.00 (0.00 – 2.32) 0.00 (0.00-4.45)
Synergistetes 0.00 (0.00 – 0.28) 0.00 (0.00 – 0.13) 0.00 (0.00 – 0.72) 0.00 (0.00-0.03)
Lentisphaerae 0.00 (0.00 – 0.20) 0.00 (0.00 – 0.00) 0.00 (0.00 – 0.93) 0.00 (0.00-0.35
Acidobacteria 0.00 (0.00 – 0.01) 0.00 (0.00 – 0.10) 0.00 (0.00 – 0.19) 0.00 (0.00-2.26)
Elusimicrobia 0.00 (0.00 – 0.00) 0.00 (0.00 – 0.00) 0.00 (0.00 – 2.04) 0.00 (0.00-0.00)

Data are presented as median and range (min-max). Kruskal-Wallis test was used for multiple comparisons between study groups.
Statistically significant differences are indicated by P < 0.05 and marked in bold type. Significance: †P<0.05, ††P<0.01 comparison between
HIT, FH or FA. Abbreviations: HC, healthy controls; HIT, histamine intolerants; FH, food hypersensitives; FA, food allergy sufferers.
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Table 3. Family level - relative abundance.

Family HC [%] HIT [%] FH [%] FA [%]

Bacteroidaceae 32.24 (10.61–58.06) 52.17 (3.82–76.56) 42.50 (3.04–75.28) 22.75 (0.03–77.76)
Ruminococcaceae 15.74 (8.83–20.91) 5.78 (1.44–20.52) 9.31 (1.27–26.26) 13.90 (3.79–25.85)
Lachnospiraceae 9.94 (2.79–17.24) 10.03 (3.52–16.04) 5.14 (1.15–17.19) 7.09 (2.07–20.86)
Porphyromonadaceae 9.40.(2.75–16.99) 7.24 (0.05–16.08) 6.22 (2.54–22.57) 8.18 (2.04–30.51)
Rikenellaceae 5.84 (1.36–14.05) 2.77 (0.00–9.51) 4.13 (0.09–14.68) 5.27 (0.02–21.57)
Oscillospiraceae 3.43 (0.56–16.35) 1.10 (0.11–4.29) 1.12 (0.04–8.46) 1.13 (0.03–11.84)
Veillonellaceae 1.88 (0.01–5.51) 2.99 (0.00–7.62) 1.24 (0.01–9.74) 2.14 (0.39–8.93)
Eubacteriaceae 1.45 (0.23–4.39) 1.23 (0.05–12.16) 0.70 (0.04–4.11) 0.90 (0.06–3.90)
Prevotellaceae 1.13 (0.00–47.46) 0.03 (0.01–13.93) 0.08 (0.00–63.35) 3.09 (0.01–69.12)
Sutterellaceae 0.52 (0.00–12.11) 1.02 (0.00–33.65) 0.96 (0.00–7.20) 0.68 (0.00–3.52)
Clostridiaceae 0.48 (0.01–4.79) 0.06 (0.00–0.79) 0.14 (0.01–9.78) 0.22 (0.01–4.99)
Desulfovibrionaceae 0.37 (0.00–2.31) 0.27 (0.03–0.90) 0.31 (0.00–1.68) 0.45 (0.00–3.39)
Hyphomicrobiaceae 0.37 (0.07–1.72) 0.44 (0.07–1.38) 0.41 (0.01–7.13) 0.56 (0.01–8.00)
Acidaminococcaceae 0.32 (0.00–3.87) 0.00 (0.00–5.54) 1.54 (0.00–29.46) 0.002 (0.00–9.92)
Bifidobacteriaceae 0.30 (0.02–1.58)*,** 0.02 (0.00–6.65)* 0.09 (0.00–3.86)* 0.06 (0.00–2.43)**
Coriobacteriaceae 0.20 (0.04–1.74) 0.09 (0.01–0.72) 0.06 (0.01–1.36) 0.07 (0.00–0.50)
Lactobacillaceae 0.14 (0.00–1.19) 0.17 (0.00–1.36) 0.10 (0.00–5.27) 0.06 (0.00–1.14)
Pasteurellaceae 0.12 (0.00–1.13)* 0.02 (0.00–0.11) 0.003 (0.00–1.62)*,† 0.025 (0.00–0.93)†
Peptostreptococcaceae 0.11 (0.01–0.28) 0.18 (0.00–9.09) 0.06 (0.00–13.68) 0.20 (0.03–2.34)
Peptococcaceae 0.10 (0.00–0.84) 0.02 (0.00–0.53) 0.05 (0.00–0.92) 0.08 (0.00–0.58)
Erysipelotrichaceae 0.08 (0.02–0.33) 0.05 (0.00–0.81) 0.06 (0.01–0.45)†† 0.16 (0.03–3.34)††
Clostridiales Family
0.06 (0.02–0.42) 0.06 (0.00–0.21) 0.21 (0.00–0.27) 0.06 (0.00–0.400)
XIII, Incertae Sedis
Streptococcaceae 0.06 (0.00–1.10) 0.07 (0.00–0.43) 0.03 (0.00–1.55) 0.03 (0.00–0.35)
Enterobacteriaceae 0.05 (0.00–2.18) 0.17 (0.00–6.39) 0.05 (0.00–11.54) 0.11 (0.00–9.05)
Clostridiales Family XII,
0.02 (0.00–0.34) 0.05 (0.00–1.69) 0.02 (0.00–12.37) 0.03 (0.00–0.85)
Incertae Sedis
Verrucomicrobiaceae 0.025 (0.00–2.39) 0.00 (0.00–0.07) 0.16 (0.00–16.51) 0.08 (0.00–3.06)
Graciibacteraceae 0.009 (0.00–0.03) 0.00 (0.00–0.55) 0.00 (0.00–0.10) 0.008 (0.00–0.25)
Bdellovibrionaceae 0.00 (0.00–1.37) 0.001 (0.00–0.37) 0.00 (0.00–3.27) 0.00 (0.00–2.83)
Comamonadaceae 0.00 (0.00–1.31) 0.00 (0.00–1.19) 0.002 (0.00–1.98) 0.002 (0.00–3.94)
Sphingobacteriaceae 0.00 (0.00–1.31) 0.00 (0.00–0.01) 0.00 (0.00–0.14) 0.00 (0.00–8.22)
Rhodospirillaceae 0.00 (0.00–0.96) 0.00 (0.00–5.33) 0.004 (0.00–2.41) 0.23 (0.00–3.66)
Spiroplasmataceae 0.00 (0.00–0.92) 0.00 (0.00–0.00) 0.00 (0.00–0.15) 0.00 (0.00–0.63)
Acholeplasmataceae 0.00 (0.00–0.42) 0.00 (0.00–0.00) 0.00 (0.00–1.54) 0.00 (0.00–3.99)
Synergistaceae 0.00 (0.00–0.28) 0.00 (0.00–0.13) 0.00 (0.00–0.72) 0.00 (0.00–0.03)
Anaeroplasmataceae 0.00 (0.00–0.21) 0.00 (0.00–0.00) 0.00 (0.00–0.80) 0.00 (0.00–0.11)
Victivallaceae 0.00 (0.00–0.20) 0.00 (0.00–0.00) 0.00 (0.00–0.94) 0.00 (0.00–0.35)
Oxalobacteraceae 0.00 (0.00–0.14) 0.00 (0.00–0.20) 0.00 (0.00–0.12) 0.00 (0.00–0.11)
Desulfobacteraceae 0.00 (0.00–0.05) 0.00 (0.00–0.00) 0.00 (0.00–0.35) 0.00 (0.00–0.12)
Flavobacteriaceae 0.00 (0.00–0.03) 0.00 (0.00–0.00) 0.00 (0.00–0.98) 0.00 (0.00–0.19)
Elusimicrobiaceae 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–2.08) 0.00 (0.00–0.00)
Marinilabiaceae 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–1.74) 0.00 (0.00–0.00)
Succinivibrionaceae 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–0.63) 0.00 (0.00–0.08)
Puniceicoccaceae 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–0.36) 0.00 (0.00–0.26
Data are presented as median and range (min–max) in %.
Kruskal-Wallis test was used for multiple comparisons between study groups. Statistically significant differences are indicated by P < 0.05
and marked in bold type. Significance: *P < 0.05; **P < 0.01, comparison HC to HIT, FH or FA; †P < 0.05, ††P < 0.01, comparison between
FH and FA. Abbreviations: HC, healthy controls; HIT, histamine intolerants; FH, food hypersensitives; FA, food allergy sufferers.

The most abundant bacterial families found in all study On family level, the HC group harbored a significant higher
groups were Bacteroidaceae (38.7%), Ruminococcaceae proportion of Bifidobacteriaceae [0.30% (0.02 – 1.58%)]
(11.2%), Lachnospiraceae (8.1%), Porphyromonadaceae (7.8%), compared to the HIT [0.02% (0.00 – 6.65%); P = 0.036], FH
Rikenellaceae (4.6%) and Veillonellaceae (2.1%). Moreover, the [0.09% (0.00 – 3.86%; P = 0.027)] and FA [0.06% (0.00 – 2.43%);
detailed analysis of bacterial families revealed significant P = 0.007] group (Fig. 3, Table 3).
differences in the proportions of Bifidobacteriaceae (class The percentage of Erysipelotrichaceae was significantly
Actinobacteria; P = 0.050), Erysipelotrichaceae (class elevated in the FA group in comparison to FH group [0.16%
Erysipelotrichia; P = 0.018) and Pasteurellaceae (class (0.03 – 3.34%) versus 0.06% (0.01 – 0.45%); P = 0.012]. HC and
Gammaproteobacteria; P = 0.031) between study groups. FA group showed highest proportions of Pasteurellaceae [HC
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Fig. 3. Differences on family level between study groups. Fig. 3a shows significantly highest relative abundance of the family
Bifidobacteriaceae in the HC group compared to the HIT (P = 0.036), FH (P = 0.027) and FA group (P = 0.007). Fig. 3b shows highest
abundance of the family Erysipelotrichaceae in the FA group with significance compared to the FH group (P = 0.012). Fig. 3c displays
Pasteurellaceae with significant highest levels for the HC and the FA group compared to the FH group (P = 0.014 and P = 0.021).
Kruskal-Wallis test was used for multiple comparisons between study groups. Abbreviations: HC, healthy controls; HIT, histamine
intolerants; FH, food hypersensitives; FA, food allergy sufferers. Significance: *P < 0.05 and **P < 0.01.

0.12% (0.00 – 1.13%) and FA 0.03% (0.00 – 0.93%)], reaching In contrast, bacteria of the genus Butyricimonas were
significance between HC and FH group (P = 0.014) and between significant less abundant in the HIT group [0.00% (0.00 –
FA and FH group (P = 0.021). 0.24%)] compared to the HC [0.42% (0.00 – 1.18%); P = 0.002]
The analysis on genus level, the most detailed bacterial and FH group [0.04% (0.00 – 2.20%); P = 0.046] (Fig. 4c).
classification in our analysis, revealed significant differences in Additionally, the genus Hespellia was significantly higher in the
six bacterial genera between the study groups (Fig. 4). These HC [0.03% (0.00 – 0.20%); P = 0.03] and FA [0.03% (0.00 –
include Roseburia (family Lachnospiraceae; P = 0.021), 0.12%); P = 0.013] group compared to the HIT group [0.00%
Dendrosporobacter (family Veillonellaceae; P = 0.008), (0.00 – 0.06%)] (Fig. 4d).
Butyricimonas (family Porphyromonadaceae; P = 0.026), Differences were also observed for genus Haemophilus
Haemophilus (family Pasteurellaceae; P = 0.034), Hespellia with elevated percentages for the HC [0.14% (0.00 – 1.33%)]
(family Clostridiaceae; P = 0.025), and Obesumbacterium and FA group [0.003% (0.00 – 1.76%)], which were significant
(family Enterobacteriaceae; P = 0.001) (Table 4). compared to the FH group (P = 0.015 and P = 0.025). The FA
Thereby patients of the HIT group showed elevated group [0.37% (0.00 – 2.61%)] showed a significant higher
proportions of Roseburia [0.45% (0.07 – 6.72%)] reaching abundance of Dendrosporobacter compared to the HIT [0.00%
significance compared to the FH group [0.11% (0.00 – 0.82%); (0.00 – 1.09%); P = 0.012] and FH group [0.00% (0.00 –
P = 0.004)] (Fig. 4a). The genus Obesumbacterium [0.00% (0.00 0.77%); P = 0.002].
– 1.31%)] was enriched in 2 of 8 HIT patients resulting in In accordance with the elevated proportions of
significantly higher values in the HIT group compared to all Bifidobacteriaceae on family level, the HC group showed also
other study groups (HC: P = 0.001; FH: P < 0.001; FA: P = an increased amount of the genus Bifidobacterium [0.35% (0.02
0.001) (Fig. 4b). In addition, the proportion of Veillonella was – 1.93%)] compared to the HIT [0.02% (0.00 – 12.91%)], FH
elevated in the HIT group [0.13% (0.00 – 7.66%)], but did not [0.09% (0.00 – 6.81%)] and FA group [0.07% (0.00 – 2.95%)],
reach statistical significance. even though the values did not reach significance (P = 0.052).
586

Bacterial diversity and cluster analysis FA group (HFA = 3.0 ± 0.9) showed lower diversities than the
HC group, but significantly higher values compared to the HIT
The PERMANOVA of the Shannon-Weaver-Index (H) group (P = 0.009 and P = 0.002). In accordance, the Simpson’s
revealed significant differences in bacterial α-diversity between reciprocal index confirmed the significantly lower α-diversity of
the study groups (P = 0.019) (Fig. 5a). Especially patients from the HIT group compared to all other groups (Fig. 5b).
the HIT group showed a significant lower diversity compared to On genus level, the beta-diversity of HIT patients partly
all other study groups. Healthy individuals (HC group) displayed differ from the other study groups using multidimensional
the highest α-diversity index with HHC = 3.2 ± 0.7 compared to scaling by principal coordinates analysis (PCoA) or non-metric
HHIT = 2.4 ± 1.0 (P = 0.043). The FH (HFH = 2.8 ± 0. 8) and multidimensional scaling (NMDS) (Fig. 6).

Table 4. Genus level - relative abundance.

Genus HC HIT FH FA
Bacteroides 37.99 (12.15–60.74) 65.49 (7.42–84.62) 47.55 (3.65–81.65) 34.72 (0.04–81.73)
Alistipes 6.50 (1.65–16.64) 3.14 (0.00–12.63) 4.44 (0.16–16.09) 6.94 (0.03–31.33)
Faecalibacterium 5.56 (2.35–16.09) 3.79 (0.00–6.91) 3.10 (0.00–16.68) 2.38 (0.06–11.48)
Parabacteroides 4.85 (2.38–8.33) 0.24 (0.00–11.07) 4.34 (0.02–10.14) 1.96 (0.00–18.87)
Oscillibacter 4.05 (0.59–19.94) 1.38 (0.12–5.42) 1.33 (0.06–10.28) 1.65 (0.05–13.54)
Barnesiella 2.18 (0.02–12.27) 1.98 (0.04–13.44) 1.01 (0.00–14.35) 2.42 (0.02–30.74)
Ruminococcus 1.87 (0.20–13.95) 1.03 (0.00–3.70) 1.53 (0.00–8.41) 1.46 (0.00–15.51)
Dialister 1.69 (0.00–4.08) 2.40 (0.01–7.41) 1.34 (0.00–9.88) 0.87 (0.00–9.38)
Eubacterium 1.65 (0.25–4.76) 1.50 (0.05–23.48) 0.72 (0.04–6.77) 1.02 (0.07 –4.44)
Odoribacter 0.82 (0.30–2.13) 0.27 (0.00–1.58) 0.66 (0.00–2.93) 0.80 (0.00–2.16)
Sporobacter 0.54 (0.00–4.36) 0.34 (0.00–1.00) 0.10 (0.00–4.58) 0.38 (0.00–2.04)
Gemmiger 0.45 (0.08–1.86) 0.51 (0.07–2.68) 0.43 (0.01–8.05) 0.74 (0.02–8.84)
Butyricimonas 0.42 (0.00–1.18)** 0.00 (0.00–0.24)**,† 0.04 (0.00–2.20)† 0.003 (0.00–1.46)
Blautia 0.36 (0.07–0.96) 0.24 (0.03–4.08) 0.26 (0.04–1.10) 0.28 (0.13–3.05)
Bifidobacterium 0.35 (0.02–1.93) 0.02 (0.00–12.91) 0.09 (0.00–6.81) 0.07 (0.00–2.95)
Sutterella 0.34 (0.00–2.01) 0.00 (0.00–0.15) 0.006 (0.00–2.65) 0.00 (0.00–4.26)
Bilophila 0.28 (0.00–2.12) 0.005 (0.00–0.96) 0.14 (0.00–1.26) 0.17 (0.00–2.11)
Coprococcus 0.28 (0.01–1.01) 0.13 (0.01–4.35) 0.13 (0.00–3.27) 0.18 (0.01–1.2)
Prevotella 0.25 (0.00–51.46) 0.02 (0.01–16.23) 0.08 (0.00–76.14) 0.85 (0.01–69.41)
Alkaliphilus 0.21 (0.00–0.41) 0.002 (0.00–0.49) 0.007 (0.00–7.59) 0.14 (0.00–3.88)
Lactobacillus 0.16 (0.00–1.43) 0.23 (0.00–1.43) 0.10 (0.00–9.28) 0.07 (0.00–1.50)
Roseburia 0.14 (0.00–1.91) 0.45 (0.07–6.72)†† 0.11 (0.00–0.82)†† 0.27 (0.00–1.32)
Haemophilus 0.14 (0.00–1.33)* 0.02 (0.00–0.14) 0.003 (0.00–1.76)*,† 0.03 (0.00–1.01)†
Dorea 0.14 (0.02–0.47) 0.06 (0.00–2.15) 0.05 (0.00–2.16) 0.09 (0.00–2.39)
Thermotalea 0.14 (0.00–0.17) 0.00 (0.00–0.10) 0.00 (0.00–10.04) 0.003 (0.00–0.14)
Veillonella 0.12 (0.00–0.98) 0.13 (0.00–7.66) 0.04 (0.00–2.11) 0.06 (0.00–2.05)
Desulfotomaculum 0.09 (0.00–0.69) 0.02 (0.00–0.40) 0.04 (0.00–1.16) 0.05 (0.00–0.63)
Collinsella 0.09 (0.02–0.66) 0.003 (0.00–0.74) 0.03 (0.00–1.51) 0.06 (0.00–0.55)
Anaerovorax 0.07 (0.01–0.44) 0.07 (0.00–0.20) 0.02 (0.00–0.29) 0.06 (0.00–0.46)
Pseudoflavonifractor 0.07 (0.01–0.29) 0.09 (0.00–0.39) 0.07 (0.01–0.10) 0.10 (0.00–0.62)
Anaerotruncus 0.07 (0.00–0.19) 0.05 (0.00–0.37) 0.05 (0.00–1.26) 0.06 (0.00–1.48)
Streptococcus 0.06 (0.01–1.32) 0.09 (0.00–0.47) 0.03 (0.00–1.98) 0.02 (0.00–0.37)
Anaerostipes 0.05 (0.01–0.45) 0.10 (0.02–0.67) 0.05 (0.00–0.37) 0.06 (0.00–0.49)
Dendrosporobacter 0.03 (0.00–2.26) 0.00 (0.00–1.09)† 0.00 (0.00–0.77)†† 0.37 (0.00–2.61)†,††
Acidaminobacter 0.03 (0.00–0.41) 0.06 (0.00–3.28) 0.02 (0.00–15.09) 0.04 (0.00–0.80
Hespellia 0.03 (0.00–0.20)* 0.00 (0.00–0.06)*,† 0.014 (0.00–0.09)† 0.03 (0.00–0.12)†,†
Clostridium 0.02 (0.00–1.82) 0.01 (0.00–0.10) 0.01 (0.00–1.36) 0.04 (0.00–2.39)
Acetanaerobacterium 0.02 (0.00–1.51) 0.00 (0.00–0.67) 0.01 (0.00–1.56) 0.015 (0.00–2.28)
Papillibacter 0.02 (0.00–0.26) 0.01 (0.00–0.07) 0.006 (0.00–0.85) 0.02 (0.00–1.23)
Peptococcus 0.02 (0.00–0.09) 0.00 (0.00–0.17) 0.00 (0.00–0.20) 0.01 (0.00–0.47)
Desulfovibrio 0.01 (0.00–2.79) 0.07 (0.01–1.09) 0.02 (0.00–1.79) 0.12 (0.00–3.94)
Enterorhabdus 0.01 (0.00–1.05) 0.00 (0.00–0.05) 0.00 (0.00–0.14) 0.00 (0.00–0.30)
Adlercreutzia 0.01 (0.00–0.66) 0.02 (0.00–0.27) 0.007 (0.00–0.17) 0.014 (0.00–0.21)
Turicibacter 0.01 (0.00–0.22) 0.002 (0.00–0.07) 0.00 (0.00–0.34) 0.003 (0.00–0.24)
Anaerofilum 0.01 (0.00–0.09) 0.005 (0.00–0.06) 0.015 (0.00–0.16) 0.01 (0.00–0.10)
Filifactor 0.01 (0.00–0.08) 0.008 (0.00–0.38) 0.003 (0.00–0.27) 0.009 (0.00–0.33)
Lutispora 0.01 (0.00–0.05) 0.002 (0.00–0.07) 0.006 (0.00–2.88) 0.02 (0.00–2.20)
Hydrogenoanaerobacterium 0.01 (0.00–0.04) 0.011 (0.00–0.10) 0.009 (0.00–0.09) 0.015 (0.00–0.17)
Gracilibacter 0.01 (0.00–0.03) 0.00 (0.00–0.73) 0.00 (0.00–0.13) 0.01 (0.00–0.33)
Akkermansia 0.00 (0.00–5.04) 0.00 (0.00–0.13) 0.18 (0.00–17.02) 0.09 (0.00–3.48)
 587

Paraprevotella 0.00 (0.00–5.04) 0.00 (0.00–0.00) 0.00 (0.00–3.73) 0.00 (0.00–13.33)

Phascolarctobacterium 0.00 (0.00–4.65) 0.00 (0.00–7.36) 1.74 (0.00–31.49) 0.003 (0.00–10.29)
Robinsoniella 0.00 (0.00–4.60) 0.00 (0.00–0.00) 0.00 (0.00–4.52) 0.00 (0.00–0.99)
Geosporobacter 0.00 (0.00–3.55) 0.00 (0.00–0.34) 0.00 (0.00–4.06) 0.003 (0.00–2.13)
Acidaminococcus 0.00 (0.00–1.74) 0.00 (0.00–0.00) 0.00 (0.00–0.05) 0.00 (0.00–0.13)
Vampirovibrio 0.00 (0.00–1.64) 0.001 (0.00–0.43) 0.00 (0.00–3.66) 0.00 (0.00–3.24)
Schlegelella 0.00 (0.00–1.57) 0.00 (0.00–1.32) 0.00 (0.00–2.26) 0.00 (0.00–4.60)
Insolitispirillum 0.00 (0.00–1.10) 0.00 (0.00–7.08) 0.00 (0.00–2.69) 0.03 (0.00–4.09)
Spiroplasma 0.00 (0.00–1.06) 0.00 (0.00–0.00) 0.00 (0.00–0.16) 0.00 (0.00–0.72)
Olivibacter 0.00 (0.00–0.80) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–4.32)
Parapedobacter 0.00 (0.00–0.75) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–5.26)
Fastidiosipila 0.00 (0.00–0.49) 0.00 (0.00–0.00) 0.00 (0.00–0.19) 0.00 (0.00–0.27)
Acholeplasma 0.00 (0.00–0.46) 0.00 (0.00–0.00) 0.00 (0.00–2.06) 0.00 (0.00–4.56)
Paraeggerthella 0.00 (0.00–0.42) 0.00 (0.00–0.07) 0.00 (0.00–0.05) 0.00 (0.00–0.01)
Victivallis 0.00 (0.00–0.25) 0.00 (0.00–0.00) 0.00 (0.00–1.26) 0.00 (0.00–0.46)
Cloacibacillus 0.00 (0.00–0.24) 0.00 (0.00–0.15) 0.00 (0.00–0.67) 0.00 (0.00–0.02)
Anaeroplasma 0.00 (0.00–0.24) 0.00 (0.00–0.00) 0.00 (0.00–1.06) 0.00 (0.00–0.00)
Caloramator 0.00 (0.00–0.23) 0.00 (0.00–0.04) 0.00 (0.00–0.17) 0.003 (0.00–0.20)
Pelotomaculum 0.00 (0.00–0.21) 0.00 (0.00–0.00) 0.00 (0.00–0.04) 0.003 (0.00–0.04)
Alkalibaculum 0.00 (0.00–0.18) 0.00 (0.00–0.13) 0.002 (0.00–0.27) 0.02 (0.00–0.28)
Oxalobacter 0.00 (0.00–0.16) 0.00 (0.00–0.24) 0.00 (0.00–0.14) 0.00 (0.00–0.12)
Erysipelothrix 0.00 (0.00–0.16) 0.00 (0.00–0.11) 0.00 (0.00–0.01) 0.00 (0.00–0.10)
Roseateles 0.00 (0.00–0.15) 0.00 (0.00–0.00) 0.00 (0.00–0.58) 0.00 (0.00–0.01)
Ethanoligenens 0.00 (0.00–0.13) 0.00 (0.00–0.12) 0.005 (0.00–0.63) 0.003 (0.00–0.74)
Rhodospirillum 0.00 (0.00–0.12) 0.00 (0.00–0.28) 0.00 (0.00–1.10) 0.00 (0.00–0.95)
Paenibacillus 0.00 (0.00–0.12) 0.00 (0.00–0.00) 0.00 (0.00–0.18) 0.00 (0.00–0.13)
Slackia 0.00 (0.00–0.08) 0.00 (0.00–0.09) 0.00 (0.00–0.17) 0.00 (0.00–0.17)
Desulfonema 0.00 (0.00–0.06) 0.00 (0.00–0.00) 0.00 (0.00–0.39) 0.00 (0.00–0.14)
Mitsuokella 0.00 (0.00–0.05) 0.00 (0.00–0.01) 0.00 (0.00–0.20) 0.003 (0.00–0.71)
Capnocytophaga 0.00 (0.00–0.04) 0.00 (0.00–0.00) 0.00 (0.00–1.19) 0.00 (0.00–0.01)
Tindallia 0.00 (0.00–0.02) 0.00 (0.00–0.20) 0.00 (0.00–1.71) 0.00 (0.00–0.03)
Acetivibrio 0.00 (0.00–0.02) 0.00 (0.00–0.15) 0.00 (0.00–1.27) 0.00 (0.00–0.23)
Obesumbacterium 0.00 (0.00–0.00)** 0.00 (0.00–1.31)**,††,††† 0.00 (0.00–0.00)††† 0.00 (0.00–0.00)††
Elusimicrobium 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–2.78) 0.00 (0.00–0.00)
Anaerophaga 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–2.31) 0.00 (0.00–0.00)
Megamonas 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–1.29) 0.00 (0.00–1.29)
Succinivibrio 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–0.84) 0.00 (0.00–0.00)
Coraliomargarita 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–0.48) 0.00 (0.00–0.32)
Enterobacter 0.00 (0.00–0.00) 0.00 (0.00–0.07) 0.00 (0.00–0.18) 0.00 (0.00–1.51)
Selenomonas 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–2.64)
Data presented as median and range (min-max). Kruskal-Wallis test was used for multiple comparisons between study groups.
Statistically significant differences are indicated by P < 0.05 and marked in bold type. Significance: *P<0.05; **P<0.01, comparison
HC to HIT, FH or FA; †P<0.05; ††P<0.01; †††P<0.001 comparison between HIT, FH or FA. Abbreviations: HC, healthy controls; HIT,
histamine intolerants; FH, food hypersensitives; FA, food allergy sufferers.

Hierarchical clustering using Euclidean distance studied short-chain fatty acids, including butyrate, acetate and
measurement and Ward’s method did not reveal any cluster propionate (24), also the biogenic amine histamine is produced
building of study groups considering bacterial distribution on by intestinal bacteria (23, 25). Several bacterial strains possess
family level (Fig. 7). the enzyme histidine decarboxylase and are able to produce
histamine by decarboxylation of histidine (23). Most of these
strains were found in foods, mainly fermented products like
DISCUSSION cheese, meat, sauerkraut, wine or beer (26, 27).
Pugin et al. were able to isolate the histamine-producing
To our knowledge this study provides the first detailed strains Morganella morganii and Lactobacillus vaginalis from
microbiome analysis of patients with histamine intolerance. It human feces (28). The gram-negative Morganella morganii
reveals significant differences in the bacterial pattern from belongs to the family of Enterobacteriaceae of the phylum
patients with histamine intolerances, and patients with other Proteobacteria and was already described in former studies for its
food intolerances or allergies in comparison to healthy controls. histamine-secreting properties (29). Even though our study
It is generally known, that the intestinal microbiome has a huge patients showed no significant differences in the relative
influence on immunological processes of the host. Thereby, not abundance of Enterobacteriaceae, they were increased in
only the taxonomic diversity of bacteria affects the immune histamine intolerant patients with 0.17%. Moreover, on phylum
tolerance, but also bacteria derived metabolites have an level, the Proteobacteria were elevated in the HIT group. This is
important effect on human health (22, 23). Besides the well- of special interest, since several studies suggested an increase in
588

Genus Roseburia Genus Obesumbacterium Genus Butyricimonas

Genus Hespellia Genus Haemophilus Genus Dendrosporobacter

Fig. 4. Differences on genus level between study groups. Fig. 4a shows a significantly higher relative abundance of Roseburia in the
HIT group compared to the FH group (P = 0.004). Fig. 4b shows the significantly highest abundance of Obesumbacterium in the HIT
group compared to HC (P = 0.001), FH (P < 0.001) and FA (P = 0.001). Fig. 4c shows an elevated abundance of Butyricimonas in the
HC and FH group and an absence of this genus in the HIT group. Fig. 4d shows significantly higher proportions of Hespellia in the
HC compared to the HIT group (P = 0.03) and FA showed significant higher proportions compared to FH and HIT group (P = 0.034
and P = 0.013). Fig. 4e shows a significantly elevated abundance of Haemophilus in the HC and FA group compared to the FH group
(HC: P = 0.015 and FA: P = 0.025). Fig. 4f shows a significantly elevated abundance of Dendrosporobacter in the FA group, reaching
significance compared to the HIT (P = 0.012) and the FH group (P = 0.002). Kruskal-Wallis test was used for multiple comparisons
between study groups. Abbreviations: HC, healthy controls; HIT, histamine intolerants; FH, food hypersensitives; FA, food allergy
sufferers. Significance: *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 5. Alpha-diversity of study groups. Fig. 5a shows α-diversity using Shannon-Weaver-Index (SWI). HIT patients showed
significantly lower SWI compared to HC (P = 0.017), FH (P = 0.009) and FA (P = 0.002). Fig. 5b shows α-diversity using Simpson’s
F
reciprocal index (SI). HIT patients showed significantly lower (SI) compared to HC (P = 0.027), FH (P = 0.022) and FA (P = 0.006).
Multiple comparisons between study groups were calculated by PERMANOVA. Abbreviations: HC, healthy controls; HIT, histamine
intolerants; FH, food hypersensitives; FA, food allergy sufferers. Significances: *P < 0.05; **P < 0.01.

Proteobacteria as a sign for dysbioses (30). Interestingly, a inflammation may cause an epithelial dysfunction and increase
dysregulation of the innate immune response was suggested to the oxygen levels in the colon. This promotes the growth of
A
promote the intestinal overgrowth with Proteobacteria resulting facultative anaerobic bacteria, e.g. various species of
in a low-grade intestinal inflammation (30, 31). An intestinal Proteobacteria, that compete against obligate anaerobic bacteria
 589

Fig. 6. Beta-diversity of study

groups. Fig. 6a shows 3D
ordination plot based on
principal coordinate analysis
plot. Fig. 6b shows 2D
ordination plot based on non-
metric multidimensional
scaling. For beta-diversity
analysis unweighted UniFrac
distance was calculated based
on genus level. Abbreviations:
HC, healthy controls; HIT,
histamine intolerance; FH,
food hypersensitivity; FA,
food allergy.

(32) comprising beneficial ones like Bifidobacteria. Therefore, an histamine formation and secretion (8). Interestingly, data of
increase in Proteobacteria is also suggested as a marker for murine models and patients with inflammatory bowel disease
epithelial dysfunction (32). An overgrowth of this phylum was suggest a protective effect of HDC-positive bacteria like
already described in patients with inflammatory bowel disease Lactobacillus reuteri on developing colorectal neoplasia by the
(33, 34). Also patients with post-infectious or diarrhea- suppression of chronic intestinal inflammation (37).
predominant irritable bowel syndrome showed an increase of However, none of our study participants showed a
Proteobacteria in comparison to healthy controls (35, 36). In significant increase in Lactobacillus. Thus, we could not detect a
healthy persons, an intestinal abundance of Proteobacteria is significantly higher abundance of histamine-producing bacteria
described between 2.5 – 4.6% (30). The increased abundance of in histamine intolerant patients, at least on phylum, family or
Proteobacteria (5.4%, range 1.3 to 34.6%) in our study patients genus level. A more precise characterization of bacteria on
with histamine intolerance compared to healthy controls indicates species level, especially of the phylum Proteobacteria, may
a dysbiosis and/or altered epithelial function in this patient group. reveal more detailed results (38) and should be examined in the
Within the Lactobacillus family some species display amino future. The influence of bacterial derived histamine as cause of
acid decarboxylase activity, e.g. Lactobacillus casei or an elevated intestinal histamine exposure in histamine intolerant
Lactobacillus delbrueckii subsp. bulgaricus, resulting in subjects is therefore questionable.
590

Fig. 7. Cluster analysis. Dendrogram displays cluster analysis with Ward method on family level. Abbreviations: HC, healthy controls
(dark blue); HIT, histamine intolerance (light blue); FH, food hypersensitivity (green); FA, food allergy.

Nevertheless, a dysbiosis may promote a mucosal Decreased levels of colonic butyrate could lead to an impaired
inflammation in the gut. Since the histamine-degrading DAO is barrier function and were already noticed in inflammatory bowel
synthesised by mature enterocytes and stored in the mucosal diseases (45-47). Maybe the lower abundance of these bacteria in
epithelial cells (39), a disruption of these cells caused by our histamine intolerant patients may exert some unfavorable
inflammation may also contribute to a reduced DAO synthesis. effects on patient’s health. But, even though the health promoting
This could lead to a reduced degradation of exogenous histamine effects of butyrate and its important role as energy source for
and results in increased endogenous histamine levels, which enterocytes is well accepted, there are also contrary reports. In
cause the typical symptoms of histamine intolerance. Since we this context, increased butyrate levels were described in patients
did not examine the intestinal inflammation status, future studies
A with self-reported food hypersensitivity and elevated levels of
should include the analyses of molecular inflammation markers, butyrate also induced visceral hypersensitivity in rats (48, 49).
e.g. of intestinal mucosa samples, to evaluate the association In comparison to all other study groups, the healthy
between dysbiosis and intestinal inflammation. persons of our study showed the highest abundance of the
However, we observed a significantly higher abundance of family Bifidobacteriaceae with a median of 0.3%. This
the bacterial genera Roseburia in patients with histamine bacterial family harbors health-beneficial species (44).
intolerance compared to healthy controls and the other food Bifidobacteriaceae decrease the intestinal pH value through
intolerant subjects. Interestingly, Roseburia is known to produce production of acetic and lactic acids and thus inhibit the growth
butyrate and health-promoting effects were attributed to this of potential pathogenic bacteria and block their adhesion to the
bacterial genus (40). In accordance, a reduced abundance of intestinal mucosa (44). Also an alleviation of gastrointestinal
Roseburia spp. was found in patients with ulcerative colitis (41) symptoms (e.g. diarrhea, constipation) and immune stimulating
or in patients with chronic kidney disease (42). Different species properties were described (50, 51), and reduced numbers of
of Roseburia produce short-chain fatty acids by metabolizing Bifidobacteria were already observed in several disorders
indigestible carbohydrates, for example starch, inulin or xylan, in including allergies, irritable bowel syndrome and inflammatory
the human colon (43). The increased proportions of Roseburia in bowel disease (50). An in vitro study of Hsieh et al.
the stool samples of our histamine intolerant patients could be demonstrated that the supplementation of the probiotic
caused by the carbohydrate and fiber-rich diet of these patients, Bifidobacterium bifidum induced an enhanced epithelial
which might exert prebiotic properties and promote the growth of function by promoting the epithelial tight junction integrity in
butyrate-producing bacteria (44). However, histamine intolerant the human intestinal epithelial cell line Caco-2 (52). This
patient showed significantly lower levels of the bacterial genus suggests a protective effect of Bifidobacterium species on gut
Butyricimonas which also belongs to the butyrate-producers. barrier, and a lack or reduced numbers of Bifidobacteria may
 591

contribute to an impaired gut barrier in different patient groups, in patients with histamine intolerance nor an enrichment of known
e.g. in our histamine intolerant patients. histamine-producing bacteria. Nevertheless, a dysbiosis in
Furthermore, we observed an increased abundance of the histamine intolerant patients may contribute to the mucosal
phylum Verrucomicrobia, especially in patients with food inflammation. This in turn could favor the development of a leaky
hypersensitivity, but very low numbers in patients with HIT. A gut and the reduction of intestinal DAO leading to elevated
high colonization with Verrucomicrobia was described in histamine levels and clinical symptoms in sensitive patients.
patients treated with a broad-spectrum antibiotic therapy (53). In addition to the necessity of a larger cohort with histamine
However, neither patients with FH nor patients of the other study intolerant patients to confirm our preliminary findings, the
groups had a recent antibiotic treatment. determination of bacterial species as well as the identification of
Interestingly, the food allergy patients of our study showed mucosa-associated bacteria might provide an even more detailed
elevated levels of the family Erysipelotrichaceae (54), which insight in the intestinal bacterial pattern in future studies.
belongs to the phylum of Firmicutes. A high abundance of this
family was described in patients with inflammatory bowel disease Clinical Trials Registration: NCT02293343.
and metabolic disease. An association between Erysipelotrichaceae
and the lipid metabolism of the host is assumed (54), but species of M. Schink and P.C. Konturek contributed equally to this work.
this family are also supposed to mediate strong immunogenic
properties and promote inflammation. In this context, a study Acknowledgements: We acknowledge the support by the H.W.
observed a positive correlation of the relative abundance of & j. Hector Foundation for funding our research. Yurdaguel Zopf
Erysipelotrichi with TNF-α (55), an important mediator of immune has received financial support for research from the STAEDTLER
and inflammatory responses, which is also involved in the process foundation, Nueremberg, Germany (Grant/Award Number: DS/eh
of allergic reactions (56), but we could not find any correlation in 35/14). The analysis of diamine oxidase activity was sponsored
our study (data not shown). and conducted by the Immundiagnostik AG, Bensheim, Germany.
Furthermore elevated levels of the phylum Firmicutes are
described in infants with food allergy (14), but we could not Conflict of interests: None declared.
confirm these findings in our study. With regard to the whole
bacterial composition, individuals that were classified as histamine
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