Intestinal microbiome dysbiosis in alcohol-dependent patients and its effect on rat behaviors

ABSTRACT Alcohol dependence (AD) is a worldwide epidemic of psychiatric disorders. Gut microbiota dysbiosis may be involved in the development of AD. Recently, the number of studies on the relationship between gut microbiota and alcohol is increasing. However, most previous studies were focused on rodent models and gut microbiota, and the data about the alteration of the gut fungi in AD patients were scarce. Convincing evidence of the gut microbiota’s role in the development of AD is lacking. Here, we investigated the characteristics of gut microbiota (bacterial) and mycobiota (fungal) dysbioses in AD patients and assessed the role of the gut microbiome on the behaviors of rats by fecal microbiota transfer, trying to illuminate the possible microbiota mechanism in AD development. We found that AD patients developed gut microbiota dysbiosis, displayed by the fact that the bacterial genus of Ruminococcaceae significantly decreased and that of Lachnospiraceae increased and the fungal genus of Saccharomyces and Kurtzmaniella obviously increased and that of Candida decreased. Alcohol consumption disturbs the gut equilibrium between bacteria and fungi. The fungal-to-bacterial species ratio was significantly decreased, and the bacterial-fungal trans-kingdom interactions were reduced. By transplanting the gut microbiota from AD patients to rats, we confirmed that the microbiota from AD patients induced the behavioral alterations associated with alcohol dependence in rats, including increased anxiety- and depression-like behaviors, reduced exploratory and recognition memory, and higher alcohol preference. Meanwhile, the microbiota from the AD patient donor upregulated the expression levels of cholecystokinin receptors in the frontal lobe, hippocampus, and cortex in rats, which might be involved in the development of alcohol dependence. Our results demonstrate that AD patients developed gut bacterial and fungal microbiota dysbioses and the gut microbiota may be involved in the development of alcohol addiction by regulating the endogenous cholecystokinin and related receptors’ expression. Our data open new avenues for adjuvant treating AD patients via administrating the intestinal microbiota. IMPORTANCE Intestinal microbiome dysbiosis is associated with psychiatric disease through the “microbiota-gut-brain” axis. Here, we revealed that there was obvious intestinal microbiome (including bacterial and fungal) dysbiosis in alcohol-dependent patients. Alcohol consumption seriously disturbs the gut equilibrium between bacteria and fungi, reduces the interactions among bacterial-fungal trans-kingdom, and increases intestinal permeability. Gut microbiota should be considered as a whole to study the development of alcohol dependence. The gut microbiome of alcohol-dependent patients increased the anxiety- and depression-like behavior in rats. The gut microbiota dysbiosis may promote the development of alcohol dependence by regulating the endogenous cholecystokinin (CCK) and related receptors. Hence, regulating the balance of gut microbiota and the endogenous CCK may be a potential strategy for reducing the risk of relapse in alcohol addiction patients.

provided by Shanghai Xinruan Information Technology Co.,Ltd.A single rat was gently placed in the corner of the chamber, and after 1 min of adaptation, all spontaneous activities were recorded for 5 min using the video-computerized tracking system.The total motion distance was used as an index of locomotor activity, while time and distance spent in the center (inner 25% of the surface area) were construed as an index of anxiety-like behavior.

Elevated plus maze test (EPM)
The EPM is another apparatus for an assessment of anxiety-like behaviors in rodents.This apparatus is elevated 76 cm from the ground plus a platform with 4 arms (2 open arms and 2 closed arms).
The rat was placed in the intersection of the 4 arms, facing an open arm, and was allowed to explore for 5 min.A video tracking system (Ethovision 11.0 XT, Noldus, Information Technology) recorded the exact track of each animal, as well as the total distance traveled and the time spent exploring each of the arms.The time spent and the number of entries into the open arms were used to assess anxiety-like behavior.

Forced swim test (FST):
FST was performed to assess depression-like behavior.Rats were placed in an inescapable transparent Plexiglas tank (20 cm x 100 cm) filled with autoclaved water (30 cm from the bottom) at 23±1°C.The test was performed in two sessions: the first session (pre-test) had a test duration of 15 min (in order to avoid the stress of the rats to affect the results of the experiment).24 h later, it was then followed by the second session (test) of 5 min.A video camera recorded each mouse for 5 minutes and manual scoring was performed by a trained experimenter who was blinded to the group allocation.Duration of immobility and latency to immobility were used to assess depression-like behavior.Water was renewed between each mouse.Before returning to their home cage, the mice were gently dried with paper towels.

Y-maze:
The Y-maze is an apparatus for assessing spatial recognition memory, which consists of three arms (one start arm and two test arms), each 45 cm in length × 10 cm in width × 29 cm in height.During the training phase, one of the test arms was blocked, and rats were allowed to explore the start arm and open test arm for 10 min. 2 h later, they were placed back in the Y-maze for 5 min, with all three arms open.The time spent in the novel arm was used as a measure of spatial recognition memory.
A preference index, a ratio of the amount of time spent in the novel test arm over the total time spent in the novel and familiar test arms, was used to measure recognition memory.

Novel object recognition
The novel object recognition test was done in the same apparatus as the open field test.Rats were first habituated to the apparatus with three sessions of 5 min each.Then rats were placed back into the arena, for the training session with two identical objects, for a period of 5 min.Objects were black-painted wooden cubes or spheres, approximately 4 cm across and high.90 min after the training session, mice were placed back into the arena, for the retention session with one familiar object the same as that was used in the training session, and one novel object (same color, different shape).The time spent interacting with the two objects was measured manually, defined as rats touching the object or sniffing the object within a distance of 2 cm.The total time spent interacting with objects was used as a measure of exploratory behavior, and the time spent interacting with the novel object compared to the familiar object was used as a measure of retention memory.A preference index, a ratio of the amount of time spent exploring the novel object over the total time spent exploring both objects, was used to measure recognition memory.

Figure
Figure S1.The location of the hospital where the samples were collected.The red dot represents the areas where the samples were collected.

Figure S2 .
Figure S2.Cladogram of LEfSe Analysis from phylum to genus level between the gut microbiota of AD patients and CT subjects.AD: Alcohol-dependent group; CT: control groups.

Figure S3 .
Figure S3.PCA score plots of metabolites in feces from AD vs CT group subjects.AD: alcoholdependent group; CT: control group, QC: quality control.

Figure S4 .
Figure S4.The results of the OPLS-DA model permutation test of the different metabolites between AD and CT groups.OPLS-DA: Orthogonal Projections to Latent Structures-Discriminant Analysis; AD: alcohol-dependent group; CT: control group.

Figure S5 .
Figure S5.The volcano plot of the differential metabolites between AD-vs CT groups.Red presents the upregulating of metabolites in the AD group vs group.Blue presents the down-regulating in the AD group.AD: alcohol-dependent group; CT: control group.

Figure S6 .
Figure S6.Quantification of the total bacterial load in the feces of rats before and after antibiotics treatment.

Figure S7 .Figure S8 .
Figure S7.The behavior of rats after FMT.(A) The total distance traveled by rats in the elevated plus maze test, and (B) in the open field test.FMT: feces microbiota transplantation, AD: alcoholdependent group, CT: control group.