Синергизм и антагонизм в микробных сообществах кишечника в организованных коллективах закрытого типа

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Аннотация

Введение. Микробиота кишечника составляет наибольшую часть всего микробиома человека. Формирование стабильной микробиоты начинается с родов, продолжая изменяться в течении жизни под действием различных экзогенных и наследственных факторов. Одними из таких внешних факторов является нахождение в закрытых организованных коллективах, где у разных лиц из-за общности быта и питания, происходит перестройка микробных сообществ кишечника. Помимо количественных и качественных изменений микробиоты, происходит и изменения во взаимоотношениях (синергизм, антагонизм, мутуализм) между микробными сообществами. Цель исследования — анализ синергических и антагонистических взаимоотношений микробных сообществ кишечника у лиц в закрытых организованных коллективах.

Материалы и методы. В группу исследования вошли 120 человек мужского пола в возрасте от 18 до 22 лет, проживавшие в пределах одного закрытого организованного коллектива на протяжении 9 месяцев. Отбирался кал для посева до начала пребывания в закрытом коллективе (1 этап), и спустя 9 месяцев после (2 этап). Идентифицированные микроорганизмы были отнесены в группу постоянной, добавочной или случайной микробиот. Для оценки связи между парами родов был рассчитан коэффициент сходства Жаккара.

Результаты. Результаты исследования показали, что синергические связи между представителями постоянной микробиоты сохраняют стабильность или усиливаются с течением времени, что в целом соответствует данным о свойствах облигатной микробиоты. Также были выявлены положительные синергические связи с добавочной микробиотой, например между Bifidobacterium spp. и порядком Lactobacillales. Синергизм данных родов способен эффективно поддерживать нормальное функционирования ЖКТ. Однако были отмечены и антагонистические взаимоотношения, особенно между некоторыми представителями добавочной и постоянной микробиоты, такими как Klebsiella spp. Такие данные могут указывать на негативное влияние некоторых микроорганизмов на микробиоту кишечника в условиях ограниченного коллектива.

Заключение. Дальнейшие исследования в этой области помогут объяснить изменения микробных сообществ в организованных коллективах и разработать стратегии для поддержания здоровой микробиоты в подобных условиях.

Полный текст

Inroduction

The gut microbiota make up the largest part of the entire human microbiome and play a crucial role in maintaining healthy homeostasis. Colonization of the gastrointestinal tract by microorganisms and formation of a stable microbiota begin with childbirth and continue to change throughout life under the influence of various external (lifestyle, diet, medications, geographical location) and inherited factors [6, 10]. One of these external factors is the presence in closed organized collectives (for example, in military units), where different individuals, due to the common way of life and changing the diet to the same type, undergo a restructuring of intestinal microbial communities [4, 5, 8, 9]. In addition to quantitative and qualitative changes in the microbiota, there are also changes in the relationships between microbial communities. Synregism and antagonism of various microorganisms can both favorably affect the physiology of the gastrointestinal tract, (for example, antagonism of the intestinal microbiota against pathogens forms colonization resistance) and contribute to the development of pathological processes (for example, the exchange of resistance genes, biofilm formation, etc.) [2, 13].

The aim of this study is to analyze the synergistic and antagonistic relationships of intestinal microbial communities in closed organized collectives.

Materials and methods

The study group included 120 people aged 18 to 22 years, male, who lived within one closed organized collective for 9 months. Feces were collected from participants for sowing before the start of their stay in a closed collective (stage 1 of the study), and 9 months after (stage 2 of the study). The study was approved by the Bioethics Committee at Samara State Medical University (protocol No. 252 dated 09/07/2022). Collection and transportation of biomaterial for microbiological research was carried out in accordance with Methodological Guidelines 4.2.2039-05 “Technique for collecting and transporting biomaterials to microbiological laboratories”. The biomaterial was sowed under anaerobic conditions using a Bactron 300-2 anaerobic station (Sheldon Manufacturing Inc., USA) on an extended range of nutrient media: MacConkey agar (HiMedia, India), Veillonella agar (HiMedia, India), Clostridium agar (Condalab, Spain), Bifidobacterium agar (HiMedia, India), Anaerobic agar (HiMedia, India), Brucella agar (HiMedia, India), Muller–Hinton agar with 5% sheep blood (HiMedia, India), chromogenic agar (HiMedia, India), Lactobacillus agar (Condalab, Spain), Saburo agar (HiMedia, India). Cultivation was carried out at a temperature of 37°C for 120 hours. The cultures were identified by MALDI-ToF mass spectrometry using a “MicroflexLT” instrument (Bruker, Germany). For all identified microorganisms, the coefficient of constancy (C) was calculated, depending on which they were assigned to the group of constant (C > 50%), additional (25% < C > 50%) or random (C < 25%) microbiota [3]. To assess the relationship between pairs of genera belonging to permanent and additional microorganisms, the Jaccard index (q) was calculated, depending on which relationship was evaluated as antagonism (q ≤ 30%), synergy (q = 30–70%) or mutualism (q ≥ 70%) [3]. Statistical analysis was carried out using the StatTech v. 4.6.3 program (developer — StatTech LLC, Russia). Categorical data were described using absolute values and percentages. Quantitative indicators with normal distribution were described using arithmetic means (M) and standard deviations (SD), 95% confidence interval limits (95% CI). In the absence of normal distribution, quantitative data were described using the median (Me) and lower and upper quartiles (Q1–Q3).

Results and discussion

As a result of the study, the permanent intestinal microbiota at the first stage included the following microorganisms: Aspergillus spp. (52.5%), Enterococcus spp. (84.2%), Escherichia spp. (100%), Lactobacillus spp. (61.7%). At the second stage, it included: Enterococcus spp. (85.8%), Escherichia spp. (100%), Klebsiella spp. (55%), Lactobacillus spp. (53.3%), Staphylococcus spp. (65%), Streptococcus spp. (53.3%).

Pairs were identified to compare the constant gut microbiota. The results of calculating the Jaccard index for pairs of constant microbiota are presented in Table 1.

 

Table 1. The results of calculating the Jaccard index for pairs of constant microbiota

Pair

Research stage

a*

b**

c***

q****

Relationship direction

Aspergillus spp. + Escherichia spp.

Stage 1

63

120

63

52.5

Synergism

Stage 2

53

120

53

44.1

Synergism

Aspergillus spp. + Enterococсus spp.

Stage 1

63

101

54

49.0

Synergism

Stage 2

53

103

46

41.8

Synergism

Aspergillus spp. + Lactobacillus spp.

Stage 1

63

74

39

39.8

Synergism

Stage 2

53

64

25

27.1

Antagonism

Escherichia spp. + Staphylococcus spp.

Stage 1

120

29

29

24.1

Antagonism

Stage 2

120

78

78

65.0

Synergism

Enterococсus spp. + Staphylococcus spp.

Stage 1

101

29

24

22.6

Antagonism

Stage 2

103

78

68

60.1

Synergism

Klebsiella spp. + Staphylococcus spp.

Stage 1

59

29

13

17.3

Antagonism

Stage 2

66

78

42

41.1

Synergism

Lactobacillus spp. + Staphylococcus spp.

Stage 1

74

29

16

18.39

Antagonism

Stage 2

64

78

49

52.69

Synergism

Staphylococcus spp. + Streptococcus spp.

Stage 1

29

40

8

13.11

Antagonism

Stage 2

78

64

45

46.39

Synergism

Note. *a — the number of subjects from whom the first microorganism was isolated; **b — the number of subjects from whom the second microorganism was isolated; ***с — the number of subjects in whom both microorganisms were isolated from the corresponding pair; ****q — Jaccard index.

 

As a result of the study, the following microorganisms were included in the additional intestinal microbiota at the first stage: Bacillus spp. (30%), Bifidobacterium spp. (43.3%), Citrobacter spp. (32.5%), Klebsiella spp. (49.2%), Lactococcus spp. (25.8%), Streptococcus spp. (33.3%). At the second stage, it included: Aspergillus spp. (44.2%), Bifidobacterium spp. (48.3%), Citrobacter spp. (25.8%), Clostridium spp. (25%), Lacticaseibacillus spp. (40.8%), Ligilactobacillus spp. (29.2%), Limosilactobacillus spp. (29.2%), Micrococcus spp. (35%), Pseudomonas spp. (25.8%).

Pairs were identified to compare the additional gut microbiota. The results of calculating the Jaccard index for pairs of additional microbiota are presented in Table 2.

 

Table 2. The results of calculating the Jaccard index for pairs of additional microbiota

Pair

Research stage

a*

b**

c***

q****

Relationship direction

Bacillus spp. + Klebsiella spp.

Stage 1

36

59

18

23.38

Synergism

Stage 2

20

66

12

16.22

Antagonism

Bifidobacterium spp. + Klebsiella spp.

Stage 1

52

59

22

24.72

Antagonism

Stage 2

58

66

37

42.53

Synergism

Bifidobacterium spp. + Ligilactobacillus spp.

Stage 1

52

0

0

0.00

Antagonism

Stage 2

58

35

22

30.99

Synergism

Bifidobacterium spp. + Limosilactobacillus spp.

Stage 1

52

0

0

0.00

Antagonism

Stage 2

58

35

22

30.99

Synergism

Bifidobacterium spp. + Streptococcus spp.

Stage 1

52

40

18

24.32

Antagonism

Stage 2

58

64

35

40.23

Synergism

Citrobacter spp. + Klebsiella spp.

Stage 1

39

59

25

34.25

Synergism

Stage 2

31

66

18

22.78

Antagonism

Klebsiella spp. + Lactococcus spp.

Stage 1

18

31

20

68.97

Synergism

Stage 2

66

22

18

25.71

Antagonism

Klebsiella spp. + Streptococcus spp.

Stage 1

18

40

24

70.59

Mutualism

Stage 2

66

64

40

44.44

Synergism

Lacticaseibacillus spp. + Limosilactobacillus spp.

Stage 1

0

0

0

0.0

Antagonism

Stage 2

49

35

20

31.2

Synergism

Ligilactobacillus spp. + Pseudomonas spp.

Stage 1

0

12

0

0.0

Antagonism

Stage 2

35

31

16

32.0

Synergism

Aspergillus spp. + Micrococcus spp.

Stage 1

63

25

10

12.8

Antagonism

Stage 2

53

42

24

33.8

Synergism

Note. *a — the number of subjects from whom the first microorganism was isolated; **b — the number of subjects from whom the second microorganism was isolated; ***с — the number of subjects in whom both microorganisms were isolated from the corresponding pair; ****q — Jaccard index.

 

The study revealed both synergistic and antagonistic relationships between representatives of the intestinal microbiota in individuals forming an organized closed-type team.

Pairs of Aspergillus spp. + Escherichia spp. and Aspergillus spp. + Enterococcus spp. at the first stage, they have a high synergistic relationship, but at the 2nd stage of the study, this relationship is suppressed and turns into an antagonistic one. It can be assumed that this is due to the pronounced negative effect of Aspergillus spp. A similar situation can be noted in the pair Aspergillus spp. + Lactobacillus spp., where synergy has turned into antagonism. For pairs of representatives of the order Enterobacterales, 100% synergy can be noted in the 2nd stage of the study in comparison with the 1st. Also worth noting is the pair Escherichia spp. + Enterococсus spp. with a coefficient of 84.1% at the 1st stage of the study, corresponding to mutualism, followed by an increase in this relationship at the 2nd stage of the study to 85.8%.

Pairs of additional gut microbiota with a high level of synergy were analyzed. Pairs of Bifidobacterium spp. + Lacticaseibacillus spp. and Bifidobacterium spp. + Ligilactobacillus spp. at the 1st stage of the study, when forming an organized closed-type team, they are defined as antagonists, but at the 2nd stage of the study, the presented pairs are defined as synergists. A similar behavior can be observed between pairs of Bifidobacterium spp. + Klebsiella spp., Bifidobacterium spp. + Streptococcus spp., Lacticaseibacillus spp. + Limosilactobacillus spp., Ligilactobacillus spp. + Pseudomonas spp.

A pair of Bacillus spp. + Klebsiella spp. at the 1st stage of the study, it was defined as synergistic, but at the 2nd stage, the transition of communication in favor of antagonism is noted. A similar situation can be observed between pairs such as Citrobacter spp. + Klebsiella spp., Klebsiella spp. + Lactococcus spp.

Thus, the results of the study showed that the synergistic relationships between representatives of the permanent microbiota remain stable or increase over time, which generally corresponds to the data on the properties of the obligate microbiota [7, 11]. Positive synergistic relationships with additional microbiota have also been identified, for example between Bifidobacterium spp. and the order of Lactobacillales. The synergy of these genera can effectively support the normal functioning of the gastrointestinal tract, which is widely used in the production of probiotics [1]. However, antagonistic relationships were also noted, especially between some representatives of the additional and permanent microbiota, such as Klebsiella spp. In recent years, the ability to antagonize the latter has been widely discussed in the scientific community [12]. Such data may indicate a negative effect of certain microorganisms on the intestinal microbiota in closed collectives.

Additional data are needed to better understand the synergistic and antagonistic relationships between representatives of the gut microbiota. Further research in this area will help explain the dynamics of changes in microbial communities in organized collectives and develop strategies for maintaining a healthy microbiota in such conditions.

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Об авторах

А. В. Ермолаев

ФГБОУ ВО Самарский государственный медицинский университет Минздрава России

Email: k.a.kayumov@samsmu.ru

ассистент кафедры общей гигиены

Россия, Самара

К. А. Каюмов

ФГБОУ ВО Самарский государственный медицинский университет Минздрава России

Автор, ответственный за переписку.
Email: k.a.kayumov@samsmu.ru

специалист Научно-образовательного профессионального центра генетических и лабораторных технологий

Россия, Самара

А. В. Лямин

ФГБОУ ВО Самарский государственный медицинский университет Минздрава России

Email: k.a.kayumov@samsmu.ru

д.м.н., доцент, директор Научно-образовательного профессионального центра генетических и лабораторных технологий 

Россия, Самара

Д. О. Горбачев

ФГБОУ ВО Самарский государственный медицинский университет Минздрава России

Email: k.a.kayumov@samsmu.ru

д.м.н., доцент, заведующий кафедрой общей гигиены

Россия, Самара

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