Fecal microbiota characterization in a swine model undergoing experimental bariatric surgery sand clock with gastro-jejunal bypass

[Caracterização da microbiota fecal em modelo suíno submetido à cirurgia bariátrica experimental relógio de areia com bypass gastrojejunal]

ABSTRACT

Bariatric surgery induces a variety of changes, including changes in the composition of the gut microbiota. The investigation evaluated the effect of the experimental gastrojejunal bypass bariatric surgery hourglass (EBSSC G-YB) on the fecal bacterial microbiota in obese Landrace pigs. Twelve six-month-old animals were used, which were supplemented with energy from 2.5 months for a period of 120 days, enough time to reach the condition. Subsequently, they were intervened with the EBSSC G-YB and the changes in the microbiota were characterized, comparing an experimental group with two control groups in a period of 30 days. The result was that the microbiota of the three groups showed Proteobacteria, Firmicutes and Bacteroidetes as the ones with the highest relative abundance, however, at the end of the experiment a difference is highlighted, where the Phylum Spirochaetes was constant and more frequently in the experimental group, while for control groups 1 and 2 it was the Phylum Tenericutes. Although the differences were not significant, qualitatively there was greater diversity in the experimental group, with greater abundance of Escherichia-Shigella and Pseudohongiella, which may be useful as quantifiable biomarkers of weight loss in future research.

Keywords:
bariatric surgery; gastrointestinal microbiome; metabolic surgery; microbiota; obesity

RESUMO

A cirurgia bariátrica induz uma variedade de alterações, incluindo alterações na composição da microbiota intestinal. A investigação avaliou o efeito da cirurgia bariátrica experimental relógio de areia com bypass gastrojejunal (EBSSC G-YB) na microbiota bacteriana fecal em porcos Landrace obesos. Foram utilizados 12 animais, com seis meses de idade, os quais foram suplementados com energia a partir de 2,5 meses, por um período de 120 dias, tempo suficiente para atingir a condição. Posteriormente, foi realizada intervenção com o EBSSC G-YB e avaliadas as alterações na microbiota, comparando-se um grupo experimental com dois grupos controle, em um período de 30 dias. O resultado foi que a microbiota dos três grupos apresentou Proteobacteria, Firmicutes e Bacteroidetes como as de maior abundância relativa, porém, ao final do experimento, destaca-se uma diferença, em que o filo Spirochaetes foi constante e com maior frequência no grupo experimental, enquanto para os grupos controle 1 e 2, foi o filo Tenericutes. Embora as diferenças não tenham sido significativas, qualitativamente houve maior diversidade no grupo experimental com maior abundância de Escherichia-Shigella e Pseudohongiella, o que pode ser útil como biomarcadores quantificáveis ​​de perda de peso em pesquisas futuras.

Palavras-chave:
cirurgia bariátrica; cirurgia metabólica; microbiota; microbioma gastrointestinal; obesidade

INTRODUCTION

Obesity is the abnormal or excessive accumulation of fat that can have serious consequences for human and animal health and currently represents a growing problem (Rinonapoli et al., 2021). By 2016, WHO indicated that 1.9 billion adults over 18 years of age were overweight and by 2020 about 39 million children were overweight or obese (Rinonapoli et al., 2021); (Purdy and Shatzel, 2021).

Obesity is considered a multifactorial disease, with serious consequences such as increased bone mineral density, leading to an increased risk of fractures (derived from metabolic factors or a greater predisposition to falls) (Rinonapoli et al., 2021), systemic inflammation mediated by interleukins (such as IL-6 and IL-8) and tumor necrosis factor (TNF)-α, which contributes to leukocytosis, increased C-reactive protein and increased platelet count (Purdy and Shatzel, 2021). Additionally, it constitutes the main risk factor for several diseases such as type 2 diabetes mellitus, coronary heart disease and some types of cancer (Davoodi et al., 2013);(Powell-Wiley et al., 2021); (Fink et al., 2022).

Among the main causes of obesity is the energy imbalance between calories consumed and calories expended, derived from the excessive consumption of foods rich in fats and sugars, and an increase in physical inactivity. Since this disease is considered preventable, important efforts have been made worldwide in the generation of policies and programs aimed at healthy eating, physical activity and psychosocial support (Lum et al., 2021). Despite their implementation, these have not had sufficient impact and obesity has continued to increase in recent years, making the implementation of more effective treatments and improved prevention mechanisms a priority (Fink et al., 2022; Griffith, 2022).

Bariatric surgery helps in the reduction of body weight, so it is indicated for people with obesity and in some countries it is restricted to those for whom other weight loss measures have failed (Douglas et al., 2015). Different surgical procedures have been implemented, being sleeve gastrectomy and Roux-en-Y gastric bypass the most frequently applied because of the best results in terms of weight loss; however, dietary modifications are required in patients so that these surgeries have fewer long-term complications (Welbourn et al., 2019).

In addition to the significant reduction in body weight and fat mass, bariatric surgery produces changes in metabolic markers, such as plasma levels of adipose-derived mediators, low insulin levels, increased lipolysis, and decreased protein oxidation (Adami et al., 2019; Jabbour and Salman, 2021). Most studies have focused on evaluating the effects of bariatric surgery on body composition and metabolic changes; however, recent studies indicate the essential role of the gut microbiota in body weight loss or gain.

The intestinal bacterial microbiota plays important roles at the immunological and metabolic level of the host, including the maintenance of intestinal homeostasis, endocrine regulation, nutrient absorption, satiety regulation, among others (Ejtahed; Hasani-Ranjbar; Larijani, 2017). Studies suggest that, after bariatric surgery, significant changes in the composition and diversity of microbial communities at the intestinal level occur in the host, varying according to the type of procedure performed (Ciobârcă et al., 2020). However, the impact of surgical interventions on the modulation of nutrients and intestinal microbiota is a field still little explored.

This study aims to describe the bacterial communities in feces of an obese swine model, operated with a novel experimental bariatric surgery (EBSSC G-YB), which combines the compartmentalization of the stomach with the rapid arrival of nutrients to the last quarter of the small intestine.

MATERIALS AND METHODS

The study was conducted in accordance with the Declaration of Helsinki and was approved and supervised by the Ethics Committee of the Faculty of Veterinary Medicine and Animal Husbandry of the University of Córdoba - Colombia (Act No.009 of November 30, 2021) for animal studies.

The experimental phase included 12 castrated Landrace pigs, 6.5 months old, average weight 115 kg with signs of overweight or obesity, from two different litters of the same pig program. The animals were housed in individual pens of 2 m² each, with bars to allow contact between animals from adjacent pens. They were kept under controlled conditions with a temperature around 23°C (± 2°C), relative humidity between 45-65% and a 12 h/12 h light/dark cycle.

The animals entered the study with an average weight of 20 kg and 2.5 months of age, fed with a commercial finishing diet (PigLevante 1 orange Italcol^®); which was enriched with 18% commercial vegetable oil from the company Megaceites S.A.S. and 15% molasses (Tab. 1), to achieve the condition of obesity, based on the information reported (Hernández Hurtado et al., 2016). They were fed twice a day (07:00 and 15:00) and the amount depended on body weight following the manufacturer's instructions. At the beginning of the study, they were clinically evaluated, and blood tests were performed with confirmation of a good state of health.

Table 1
Composition of commercial diet and energy supplement (commercial vegetable oil and molasses)

Type of diet	Composition	Quantity (%)
Commercial	Minimal protein	17
	Minimal grease	5
	Maximum humidity	13
	Maximum fiber	7
	Maximum ash	8
Energy supplement	Commercial vegetable oil1	18
	Molasses2	15

• ¹
  The commercial oil provided 80 calories of fat with a per serving amount of total fat of 9g (14%).
• ²
  Molasses provided 2100kcal/kg, which for the amount added to the commercial fattening ration corresponds to 787.5kcal.

Pigs with four months of animal management were taken from 2.5 months of age and an experimental phase from 6.5 months of age with a duration of 30 days where weighing, measurements and blood sampling were made, at the beginning of the experimental phase (day 0), on day 15 and on day 30 post-surgical intervention.

Initially, 15 animals were selected, but the sample size was reduced to 12 pigs as this was the number of pigs approved by the ethics committee for the research. They were divided into three subgroups by purposive sampling, where the first control group (CG1) consisted of three animals without surgery and that continued with energy supplementation; a second control group (CG2) consisted of four animals with celiotomy and energy supplementation was withdrawn, and the experimental group (EG) consisted of the five animals with the highest weight that underwent experimental surgery without energy supplementation (Fig. 1).

Yao et al. (2019) recommend 10g of fecal matter as a representative amount of the sample, however; approximately 20 to 30g of feces/take were collected per animal at three different times during the experiment, according to the technical guidelines of the company INNOVASEG of Bogotá-Colombia, which oversaw processing the samples. The samples were kept at -90ºC in a refrigerator at the Instituto de Investigaciones Biológicas del Trópico (IIBT) until the samples were filled. The 24 samples collected were then sent refrigerated at 4°C to the laboratory for processing, where DNA was extracted for the description of bacterial communities through complete sequencing of the rRNA-16S gene (Long reads amplicon-based sequencing) using Oxford Nanopore technology.

Figure 1
Experimental design and distribution of Landrace pigs included for identification of bacterial communities according to age stage and feeding management. Where: CG1= control group one without surgery, CG2= control group two with celiotomy and EG= experimental group.

Prior to surgery, the animals were subjected to a 6-hour and 12-hour water and solid fasting periods, respectively. All surgeries were performed under general anesthesia, however, to reduce the dose used, an initial analgesia step was performed, applying intramuscularly flunixin meglumine (1.1mg/kg b.w.) and metoclopramide (0.5mg/kg b.w.). For tranquilization azaperone (4mg/kg bw) was applied and anesthesia was induced with intramuscular injection of xylazine (0.5mg/kg) and ketamine (2 mg/kg) mixture and maintained with isoflurane. Oro-tracheal intubation was performed, and oxygen was supplied throughout the procedure, and heart rate, respiratory rate, temperature, mucosal color, capillary filling time, eyeball position and pupil size were also monitored.

The experimental technique proposed is among those classified as restrictive-malabsorptive. It seeks to reduce the gastric capacity so that the pig has an early and prolonged satiety sensation with a much smaller amount of food than that usually ingested, and that the chyme reaches the last quarter of the small intestine (distal jejunum and ileum) faster. The experimental bariatric technique is not described in the text because it is part of another publication proposal. All surgeries were performed by the author of the experimental technique, who is a veterinary surgeon and junior researcher according to Colciencias categorization. The choice was made to reduce the variability of the surgical technique and maximize its success.

After surgery, pain control was managed with intramuscular injection of flunixin meglumine (1.1mg/kg b.w.), every 12 hours for 3 days. Antibiotic prophylaxis was performed by intramuscular injection of oxytetracycline (5mg/kg b.w.) every 24 hours for three days, complemented with a single intravenous dose of metronidazole (10mg/kg b.w.) during the operation. It should be noted that the pigs in the two control groups also received this treatment. As for the daily feed ration, it was divided into three portions administered every four 4 hours.

For the extraction of DNA from the stool samples collected throughout the follow-up, an initial step was carried out with mechanical disruption by beating with beads, subsequently for DNA recovery the DNA Stool DNA Isolation kit^® (Norgene, Biotek, Corp) was used, following the manufacturer's instructions. The DNA quality of the stool samples was determined with a NanoDrop® 1000 (Thermo Fischer Scientific), the samples included complied with a concentration of >20 ng/μl and an absorbance ratio of ≥1.8/2.0 at 260/280nm.

Amplicons of the 16S rRNA gene were generated using primers 27-F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492-R (5′-GGTTACCTTGTTACGACTT-3′). PCR amplification of the 16S rRNA genes was performed with the KAPA2G™ Robust HotStart ReadyMix PCR kit (Kapa Biosystems). Amplification was performed with the following PCR conditions: initial denaturation at 94 °C for 3 minutes, 30 cycles of 94 °C for 30 seconds, 48 °C for 1 minute and 72 °C for 1 minute, followed by a final extension at 72 °C for 10 minutes. For Barcoding the SQK-NBD114.96 Kit was used and for ligation the SQK-LSK110 kit (Oxford Nanopore Technologies), the amplified DNA was purified with AMPure^® XP (Beckman Coulter), the library was loaded onto the R9.4 flow cell (FLO-MIN106; Oxford Nanopore Technologies) and sequenced on the MinION™ Mk1C. Long-read sequencing was performed using the MINKNOW app. 1.11.5 (Oxford Nanopore Technologies).

For basecalling of raw Fast5 files and demultiplexing, Guppy was used to generate approved reads (FASTQ format), then the reads were filtered by removing possible chimeric reads. The average quality score of Phred was evaluated using the NanoPack tool. Finally, taxonomic assignment was performed using the Silva v132.16s database (Fujisaka et al., 2023) and for taxonomic clustering the Kraken tool (Nagaya et al., 2021) was used. Alpha diversity analyses were performed for the ASVs of each of the groups. Thus, the Abundance-based Coverage Estimator (ACE) was calculated to obtain an estimate of richness, and the Shannon and Simpson index to obtain an estimate of community diversity in the groups of interest (Bhaskaran et al., 2014; Pacheco et al., 2019). Statistical differences in richness and diversity estimates between groups were evaluated using a Mann-Whitney or Kruskall-Wallis test (p<0.05). For beta-diversity analyses, a principal coordinate analysis (PCoA) was performed with distances calculated by Bray-Curtis. Analysis of variance permutations using distance matrices (adonis) was used to evaluate significant differences in sample clustering depending on each study group. Alpha and Beta diversity analyses were performed using the R package phyloseq (Manson et al., 1995).

Taking into account the non-normal distribution of the data (Shapiro-Wilks normality test), these were presented in terms of medians together with their corresponding measures of dispersion (interquartile ranges - IQR). For all tests the statistical significance (P value) was set at ≤0.05. STATA17 software^® was employed for statistical analyses.

RESULTS

According to Akagbosu et al. (2022), bariatric surgery induces changes in digestive tract anatomy, gastric emptying, hormonal status, quantity and choice of ingested nutrients, and bile acid metabolism, which may modify the composition of the intestinal microbiota.

Sequencing of the rRNA-16S gene by means of the Oxford Nanopore platform (Leggett and Clark, 2017), generated more than 600,000 reads in the length range of 1.2 to 1.4 kb with a quality score ≥8, which were then taxonomically identified. A total of 36,626 ASVs (Amplicon Sequence Variant) belonging to the Bacteria domain were identified and assigned, being classified as 55 Phyla and 1088 bacterial genera.

The most abundant phyla throughout the follow-up were Proteobacteria, Firmicutes and Bacteroidetes; however, by the third follow-up an increase in the phylum Tenericutes was observed (Fig. 2A). At the genus level, differences in relative abundance were observed according to age, for 2.5 months (baseline) the genera Escherichia-Shigella, Succinivibrio and Christensenellaceae R-7 group were the most abundant, for the age of 6.5 months the genera Escherichia-Shigella, Pseudohongiella and Alteromonas, and finally for 7.5 months Christensenellaceae R-7 group, Escherichia-Shigella and Streptococcus (Fig. 2C).

Regarding the bacterial taxa according to the group analyzed, the results showed Proteobacteria, Firmicutes and Bacteroidetes as those with the highest relative abundance at the phylum level (Fig. 2B). However, at the end of the experiment, a higher frequency of the phylum Proteobacteria and Tenericutes was found in CG1 and CG2 and the phylum Spirochaetes in the SG (Fig. 2B).

On the other hand, the results showed differences in the abundance of some genera, according to the group analyzed, being for CG1 Streptococcus, Escherichia-Shigella, Christensenellaceae R-7 group and Ruminococcaceae UCG-014, for CG2 the genera Succinivibrio, Escherichia-Shigella, Alteromonas and Pseudohongiella, and for EG the genera Escherichia-Shigella, Christensenellaceae R-7 group, Pseudohongiella, Lachnospiraceae NK4A136 group and Treponema (Fig. 2D).

Figure 2
Bacterial composition in stool samples Landrace pigs. (A) Bar chart showing the main bacterial phyla according to follow-up (n=18). (B) Bar chart showing the main bacterial phyla according to the group analyzed (n=24). (C) Bar chart showing the main bacterial genera according to follow-up (n=18). (D) Bar chart showing the main bacterial genera according to the group analyzed (n=24).

To estimate the alpha diversity for the ASVs encountered the richness estimator (ACE) was calculated, and the Shannon Weaver and Simpson indices were calculated to estimate the diversity of the bacterial communities (Fig. 3). The diversity analysis revealed higher richness in samples from 7.5 months old animals compared to 2.5 and 6.5 month old animals (ACE, p=0.0832; Shannon, p=0.635; Simpson, p=0.3722) (Mann-Whitney, Kruskall-Wallis) (Fig. 3A). For the analyzed group, the results showed higher indices (diversity and richness) in EG animals (ACE, p=: 0.8756; Shannon, p=0.6743; Simpson, p=0.7205 (Kruskall-Wallis), compared to the control groups (Fig. 3B). However, despite the differences these indices were not statistically significant, probably due to the number of samples analyzed. To estimate the variability of the bacterial communities, beta diversity was evaluated using principal coordinates analysis (PCoA) based on Bray-Curtis differences, where the results showed a separation in the groups analyzed (Fig. 3). PERMANOVA analysis using distance matrices (adonis) showed no statistically significant differences in the distribution of centroids according to age (r2= 0.10, p= 0.24) (Fig. 3A) and group analyzed (r2=0.18, p=0.21) (Fig. 3B). Although statistical significance was not found, it is possible to observe a grouping of the controls compared to the experimental group.

Figure 3
Analysis of alpha and beta diversity of bacterial communities in fecal matter of Landrace pigs according to age and study group. (A) diversity indices and NMDS plot with respect to follow-up (n=18). (B) diversity indices and NMDS plot with respect to the analyzed group (n=24).

On the other hand, when comparing the relative abundance of the phyla according to the group evaluated, the results showed that for the SG the phyla Firmicutes and Spirochaetes were the most abundant, for CG1 it was the phylum Ternericutes, while for CG2 it was the phylum Bacteriodetes and Proteobacteria (Fig. 4); however, these differences were not significant.

Figure 4
Differences between the relative abundance of the most abundant phyla according to the group Landrace pigs evaluated at the end of the experiment.

Finally, the differences in relative abundances between the most frequent bacterial genera in the analyzed population were compared, and it was found that Escherichia-Shigella and Pseudohongiella were significantly more abundant in the SG compared to the control groups (Fig. 5).

Figure 5
Differences between the relative abundance of Escherichia-Shigella and Pseudohongiella according to the group Landrace pigs evaluated.

DISCUSSION

The porcine model and its easy handling make it an excellent biological model that facilitates the understanding of organic functioning, which can be extrapolated to other animal species, including humans. In this sense, this model is widely used in the implementation of surgical techniques including bariatric techniques (Silva Ribeiro et al., 2020) and research aimed at the description of bacterial communities (microbiota) at the intestinal level and their impact on organ physiology and phenotype. At the microbiota level, studies suggest that particular bacterial taxa contribute to metabolic processes, which could affect energy regulation in obesity (Akagbosu et al., 2022). However, considering that this condition is of multifactorial etiology, factors additional to the microbiota such as environmental or geographical, behavioral, genetic, dietary, microbial and biological factors also contribute to its etiopathogenesis, and differences in results and conclusions apart from the above may also be influenced by study designs and sample size (Akagbosu et al., 2022).

According to Akagbosu et al. (2022), reduced bacterial biodiversity at the gut level in individuals consuming a Western diet (high in fats, sugars and animal proteins) was associated with an increased incidence of obesity, coronary vascular disease and metabolic syndrome, with the obese condition being a common parameter found in pigs from the two CGs.

With respect to the microbiota, when eubiosis is present, the pig's intestine will be in optimal condition for good functioning and will be considered healthy. The characteristics of microbial dysbiosis are not completely clear; however, it has been defined as an intestinal imbalance identified by a marked decrease in the amount of obligate anaerobic (beneficial) bacteria, such as members of the Phylum Bacteroidetes and Proteobacteria, and a relative increase in the abundance of facultative anaerobic bacteria (pathobionts), where some members of the Phylum Firmicutes are found (Fenske et al., 2020). According to Fujisaka et al. (2023), dysbiosis causes the so-called "leaky gut" syndrome in which the intestinal barrier function is impaired, with increased permeability, through decreased intestinal mucus and tight junction protein content in the intestinal epithelium. With increased intestinal permeability, the bacterial cell wall component endotoxin enters circulation and causes hyperendotoxemia. The endotoxin activates Toll-like receptor (TLR) 4, which in turn promotes chronic inflammation in adipose tissue and liver and exacerbates obesity-induced insulin resistance. The inclination of the balance towards one or another bacterial phylum influences the lean or obese body condition (Fenske et al., 2020), so it is presumed that the animals in the study and in particular those of the EG, had a healthy intestine with higher indices of bacterial diversity and richness, which are important for the eubiosis status.

According to geography, the research results resemble the results of two studies cited by (Scheithauer et al., 2022) in work conducted in France who found Proteobacteria, Bacteroides and Escherichia as the most abundant Phyla and genus post Roux-en-Y gastric bypass or sleeve gastrectomy and also resemble the results advanced in Paise Netherlands by Scheithauer et al. (2022), who found Proteobacteria and Akkermansia as the most abundant post-gastric bypass Roux-en-Y phylotype and genus, but differ from other studies such as Liu et al. (2017) who found Bacteroidetes and Bifidobacterium as the most abundant in work performed in China post-gastric bypass in Roux-en-Y, and with that of (Sánchez-Alcoholado et al., 2019) who found Bacteroidetes and Akkermansia as the most abundant in work performed in Spain post Roux-en-Y gastric bypass or sleeve gastrectomy, as also differs from the results of Ciobârcă et al. (2020) who found Bacteroidetes and Fusobacteria as the most abundant phylum and genus post adjustable banding.

According to age, (Yang et al., 2022) reported that Phylum Firmicutes and Bacteroidetes were the most abundant in rectal fecal samples of piglets from Jinfen White and Mashen breeds. According to (Zhao et al., 2015); (Vigors et al., 2016); Zhang et al. (2022) the intestinal microbial richness of pigs varies significantly with age and with intestinal segment. According to age according to Zhao et al. (2015), Proteobacteria in adult pigs represents 2% of the abundance while that of Firmicutes is greater than 90%. According to the segment, the ileum of the pig is colonized mainly by Firmicutes and Proteobacteria, and the abundance of Bacteroidetes in the cecum and colon increases significantly (Liu et al., 2017; Zhang et al., 2022), however these results do not coincide with those observed in the study pigs, where it was found that the most abundant Phyla throughout the follow-up (2.5, 6.5 and 7.5 months of age) and independent of the study group were Proteobacteria, Firmicutes and Bacteroidetes; with an increase in the Phylum Tenericutes by the third follow-up. These variations in order of abundance when compared with the works cited in the paragraph, may be related to the constituents of the diet that has among its components vegetables such as corn and the supplementation used to achieve the obese condition, which could acidify the gastro-intestinal pH to levels that favored or stimulated the formation of bacteria of the phylum Proteobacteria as reported by Akagbosu et al. 2022. The non-difference between phyla could be influenced by the sample size and the follow-up time, which did not allow identifying significant changes, being advisable for future research a larger number of animals and a longer follow-up time.

When talking about diversity between groups, the experimental group showed the highest diversity, which opens the door to raise the experimental surgery as influential in the results, towards the training of Proteobacteria which, as a Phylum; is much less abundant than Firmicutes in fecal matter of adult pigs according to Zhao et al. (2015). The results also seem to agree with those of Scheithauer et al. (2022) who report post-analysis of 40 pre-RYBG and post-RYBG cases in humans, increase in proinflammatory Proteobacteria with improvement of chronic inflammation post-surgery. They substantiate the improvement based on findings of increased lipopolysaccharide (LPS) and flagellin-specific immunoglobulin A (IgA), but total fecal IgA content remained unchanged. Thus, an increase in intestinal IgA after bariatric surgery neutralized immunogenic bacteria and their components, leading to an improvement of systemic inflammation. The results of the present investigation could possibly be influenced by the consequent lower pH of chyme emptying in distal jejunum where Proteobacteria are resistant and functionally associated with a beneficial profile characterized by decreased systemic inflammation, improved glucose homeostasis and with body weight loss (Debédat et al., 2019) which were results obtained in the EG.

The results related to the increase in microbiota diversity at different taxonomic levels were similar to the result obtained by(Juárez-Fernández et al., 2021) in obese patients after bariatric surgery, in addition; they highlight a reduction of the Phylum Firmicutes compared to the same patients before the intervention; therefore, the results suggest that bariatric experimental surgery had a greater impact on the results, than the energy restriction derived from sugar and fat.

Studies such as Guo et al., 2018 have reported that bariatric surgery can induce increase in some classes of Proteobacteria that correlate with lower serum insulin concentration Guo et al., 2018 lower BMI in patients after Roux-en-Y gastric bypass according to the report of (Sánchez-Alcoholado et al., 2019). In addition, higher Proteobacteria abundance has been associated with less food addiction after bariatric surgery according to (Oduro-Donkor et al., 2020) Whereas, higher concentrations of Firmicutes which was the predominant Phylum in the two CGs, have been related to increased metabolic degradation of energy sources, resulting in increased caloric absorption and consequently higher weight gain. In this regard, increased Firmicutes abundance has been consistently associated with obesity (Crovesy et al., 2020). It should be noted that CG1 and CG2 pigs at the end of the study were still obese (detailed information in another article) with similar BMI (=0.99), while GE pigs involuted their condition from obesity to leanness with BMI going from 0.99 to 0.93, so promoting a shift towards a healthy profile (less Firmicutes more Proteobacteria) may be a determinant of the beneficial effects of experimental bariatric surgery.

At the end of the experiment, it is worth noting a difference between the groups, where the phylum Spirochaetes was constant and with higher frequency in all the animals of the SG, while for the animals of groups CG1 and CG2 it was the phylum Tenericutes. The EG results agree in part with a previous study by LI et al., 2023 and Wang et al., 2023 who reported Phylum Spirochaetes as the third most abundant of the four phyla found in healthy growing pigs fed rice bran and in the work with finishing Creole sows, respectively.

In terms of genus, in the EG there was greater bacterial diversity; led by Escherichia-Shigella (Proteobacteria), followed by Christensenellaceae R-7 group (Firmicutes), Pseudohongiella (Proteobacteria), Lachnospiraceae NK4A136 group (Firmicutes), and Treponema (Spirochaetes); for CG1 the genera Streptococcus (Firmicutes), Escherichia-Shigella (Proteobacteria), Christensenellaceae R-7 group and Ruminocococcaceae UCG-014 (Firmicutes) and for CG2 the genera Succinivibrio (Proteobacteria), Escherichia-Shigella (Proteobacteria), Alteromonas and Pseudohongiella (Proteobacteria) (Figure 2D). At the genus level, the results of this study are discordant with respect to a previous report by Wang et al., 2023 , who found Prevotella, Treponema and Lactobacillus as the most abundant genera in finishing sows, with some differences between animals in the study, explained by other characteristics such as the animal's own gene pool, breed, sex, age stage and environmental conditions such as management and feeding. According to Wang et al., 2023 say that the genus Treponema is related to low feed conversion, so it may be of particular interest and should be taken into account as a possible biomarker in future works of evaluation of the standardized bariatric technique in the research, since it is the genus not common among the three groups of the study and is present in the EG.

On the other hand, the modifying role in the intestinal microbiota achieved in the EG after surgery, mostly represented by the genus Escherichia-Shigella; was also observed in another study, where the values were higher in people after laparoscopic gastric sleeve surgery when compared to the values of people with normo-weight (Albaugh et al., 2017), further suggesting; that this entero-bacterial genus could be used as a bio-marker or possible quantifiable indicator of a biological condition or change; such as that achieved after experimental surgery.

The increase in Escherichia/Shigella genus in EG may in part agree with the results of (Akagbosu et al., 2022)), who demonstrated alterations with a tendency to increase in Proteobacteria such as E. coli in patients with gastric sleeve (GS) and Roux-en-Y bariatric surgery (RYGB). The explanation for this alteration could be associated with an increase in luminal acidity and dissolved oxygen after these procedures, as well as the ability of E. coli to efficiently harvest energy after bariatric surgery. Regarding Escherichia coli, it can synthesize the best studied enteric bacterial protein enteric protein (ClpB), and which can act as a mimetic of alpha-melanocyte stimulating hormone (αMSH) with similar anorexigenic effects. Furthermore; E. coli-derived ClpB is able to enhance PYY and GLP 1 secretion, and directly activate anorexigenic neurons and subsequently induce satiety with effect on weight loss (Arnoriaga-Rodríguez et al., 2020; Han et al., 2021) coinciding part of the above statements , with results obtained in EG pigs.

Regarding the EG, it is presumed that the distal enteric medium where the post-surgery chyme arrives may have a very low pH due to the rapid delivery of gastric contents, less buffering and less dilution due to the short enteric pathway and possibly due to the higher concentration of gastric juices in proportion to the ration consumed, which corresponded to 1/3 of that of the CG, variables that could influence the development of bacteria resistant to acidity and with the power to digest energy sources derived from poorly digested vegetables, properties that characterize Proteobacteria, among which the Escherichia/Shigella genus can be found (Akagbosu et al., 2022).

Taking into account the results of this study and previous report (Aron-Wisnewsky et al., 2019), the application of bariatric surgeries, could affect the composition of the intestinal microbiota by inducing alterations in entero-environmental and systemic factors, as well as by anatomical changes in the digestive tract (Aron-Wisnewsky et al., 2019). Furthermore, the metabolic improvement that occurs after surgery cannot be explained only by caloric control and weight loss, but also by changes in microbial communities. Although changes in the composition of the intestinal microbiota may be caused by adaptation of microorganisms to caloric control, derived from bariatric surgeries leading to a decrease in energy harvesting, by alterations in fermentation activity and subsequent sustainable weight loss (Alqahtani et al., 2023; Wang et al., 2023).

The results of this study suggest that experimental hourglass bariatric surgery with gastro-jejunal bypass had modulatory effects on gut microbiota. The statement agrees with the findings of Scheithauer et al. (2022) who state that, metabolic improvements mediated by bariatric surgery are mainly associated with immune activities that are mediated by microbial alterations and by changes in incretin secretion.

Understanding gut microbial changes will provide key information on how weight loss is modulated in the face of bariatric surgery. In addition, characterization of the gut microbiota before and after bariatric surgery may generate preliminary data for the development of therapies based on bacterial taxa that contribute to the treatment of animal and human obesity. These therapies could improve outcomes after surgery, or even decrease the need for surgery in the future by becoming a weight loss option.

Considering that bariatric experimental surgery remodeled the intestinal bacterial microbiota in a 30-day post-surgery period, it is necessary to propose future research with a larger sample size, several cut-off points in time and a longer study time. The approach is based on the concepts of recurrence/weight maintenance and transitional/permanent microbiota, which need to be identified. The statistical model would consider a prospective investigation comparing the same animals with obese condition before and after experimental surgery and another possible comparison with a control group of non-obese animals.

CONCLUSIONS

In this first study a novel bariatric surgery titled EBSSC G-YB was standardized, which according to the statistical analysis did not show significant differences between the experimental groups. However, the results suggest that the intervention could influence the bacterial communities such as the increase achieved in the Phylum Spirochaetes with higher indexes of bacterial richness and diversity and high abundance of the genus Escherichia/Shiguella and Pseudohongiella, which could be considered as intestinal biomarkers in future research.

ACKNOWLEDGMENTS

The pigs included in the experiment and their feed during the study were provided by the swine breeding program of the Faculty of Veterinary Medicine and Animal Husbandry of the University of Córdoba-Colombia.

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Publication Dates

• Publication in this collection
  23 Aug 2024
• Date of issue
  Sep-Oct 2024

History

• Received
  15 Oct 2023
• Accepted
  20 Feb 2024