To our knowledge, we report the largest case–control study comparing human obese gut microbiota to controls focusing on Archaea, Bacteroidetes, Firmicutes, Lactobacillus genus, Lactococcus lactis and B. animalis and, for the first time, we used a culture-dependent and culture-independent method to compare the Lactobacillus population at the species level between obese and normal-weighted humans. Our results confirm global alteration in obese gut microbiota with a lower level of M. smithii as already reported in the literature,11 and newly report lower levels of B. animalis, L. paracasei, L. plantarum and higher levels of L. reuteri in obese gut microbiota.

The qPCR system used in this study to detect and quantify Bacteroidetes, Firmicutes, Lactobacillus genus and M. smithii in human feces has already been evaluated and validated.12, 29 LAMVAB-selective media has also been used successfully to identify and enumerate lactobacilli from human feces.27 As in our previous study,12 we found an increase in Lactobacillus in obese patients using the same Lactobacillus genus-specific PCR system. However, we found that its sensitivity profile was heterogeneous among the Lactobacillus species found in human feces by culture (data not shown). We subsequently developed a novel Lactobacillus species-specific qPCR system targeting species associated with obesity or normal weight in our preliminary culture study, and targeting other species present in marketed probiotics products as Lactococcus lactis and B. animalis. Species-specific Lactobacillus PCR based on the Tuf gene and designed for this new study showed good reproducibility, sensitivity and specificity. However, we found significant discrepancies between culture and Lactobacillus species-specific PCR species. First, L. gasseri and L. acidophilus could not be identified in culture due to the presence of vancomycin in the LAMVAB medium. Conversely, although qPCR was much more sensitive than culture to detect selected species of Lactobacillus, we showed that the two methods were consistent for L. casei/paracasei, L. plantarum and L. reuteri. For these three Lactobacillus species, both techniques resulted in the same effect direction with human obesity gut microbiota enriched in L. reuteri, and depleted in L. casei/paracasei and L. plantarum.

The decrease of Bacteroidetes was historically the first alteration significantly associated with obesity as reported by Ley and Turnbaugh,8 in mice and in North American individuals,7, 9 and by Santacruz et al.,15 who observed overweight pregnant women in Spain. We found the same correlation in our previous study,12 and the same effect direction in the present study with the same PCR system on the whole population and after the exclusion of common subjects. Schwiertz et al.11 reported opposite results, but the methodology was objectionable because the Bacteroidetes proportion was obtained by summing Bacteroides and Prevotella genera. Other studies found no interaction between the relative or absolute abundance of Bacteroidetes and obesity.31, 32, 33

In our previous study,12 abundance of M. smithii was significantly higher in patients with anorexia but not in lean controls. In this new study, we found that M. smithii was less frequent and significantly less abundant in obese patients on the whole population and after the exclusion of common subjects. Schwiertz et al.11 using a specific qPCR for Methanobrevibacter species, found similar results in a German population. These results are in contradiction to those of Zhang et al.33 who found that Methanobacteriales was present only in obese individuals using a qPCR but only three obese vs three controls were compared.

In this study, we report an association between lower levels of B. animalis and obesity for the first time. Five studies reported a decreased number of Bifidobacterium representatives in the feces of obese subjects at the genus level.11, 13, 14, 15, 16 At the species level, Kalliomaki et al.13 using a Bifidobacterium species-specific PCR, found that Bifidobacterium longum and Bifidobacterium breve were higher in normal weight controls, but this result was not significant probably because of a small sample size. Experimental data report that administration of a B. breve strain to mice with high-fat diet-induced obesity led to a significant weight decrease.34 Administering four different Bifidobacterium strains to high-fat diet induced obese rats, Yin et al.35 reported that one strain increased body weight gain, another induced a decrease and the two other strains lead to no significant change in body weight but species were not mentioned in this study. In this way, Cani et al.36 reported that high-fat feeding was associated with higher endotoxaemia and lower Bifidobacterium species cecal content in mice. The selective increase of bifidobacteria by oligofructose, improving mucosal barrier function, significantly and positively correlated with improved glucose tolerance, glucose-induced insulin secretion and decreased endotoxaemia.

L. plantarum and L. paracasei were associated with normal weight in culture, consistent with experimental models in the literature reporting an anti-obesity effect of L. plantarum in mice.37 Other Lactobacillus strains have shown an anti-obesity effect in animals and humans similar to the L. gasseri SBT2055 (LG2055) strain in lean Zucker rats38 and in humans.39 This anti-obesity effect may be linked to the production of specific molecules that can interfere with host metabolism, such as conjugated linoleic acid (CLA) for L. plantarum or L. rhamnosus.37, 40 In vivo and in vitro analyses of physiological modifications imparted by CLA on protein and gene expression suggest that CLA exerts its delipidating effects by modulating energy expenditure, apoptosis, fatty acid oxidation, lipolysis, stromal vascular cell differentiation and lipogenesis.37 Authors who have investigated the mechanisms linking conjugated linoleic acid and anti-obesity effects have reported the upregulated expression of genes encoding uncoupling proteins (UCP-2), which could be a primary mechanism through which CLA increases energy expenditure and produces an anti-obesity effect.40

L. reuteri has been associated here with obesity. L. reuteri has been one of the most studied probiotic species especially for its ability to inhibit the growth of other potentially pathogenic microorganisms by secreting antibiotic substances such as reuterin.41 When introduced in pigs, turkeys and rats, L. reuteri led to a significant weight gain and was isolated in higher concentrations from feces after probiotic administration.42, 43, 44 The mechanism by which L. reuteri is able to support the healthy growth of these animals is not entirely understood. It is possible that L. reuteri simply serves to protect livestock against illness caused by Salmonella typhimurium and other pathogens. However, other studies have revealed that L. reuteri can also help when the growth depression is caused entirely by a lack of dietary protein and not by contagious disease.45 This raises the possibility that L. reuteri somehow improves the intestines’ ability to absorb and process nutrients, and increase food conversion.46

As a theoretical basis for the causal link between the gut microbiota alterations and obesity, several mechanisms have been suggested. First, the gut microbiota could interact with weight regulation by hydrolysis of indigestible polysaccharides to monosaccharides easily absorbable activating lipoprotein lipase. Consequently, glucose is rapidly absorbed producing substantial elevations in serum glucose and insulin, both factors that trigger lipogenesis and fatty acids excessively stored with de novo synthesis of triglycerides derived from liver, these two phenomena causing weight gain.47 Second, the composition of gut microbiota has been shown to selectively suppress the angiopoietin-like protein 4/fasting-induced adipose factor in the intestinal epithelium, known as a circulating lipoprotein lipase inhibitor and regulator of peripheral lipid and glucose metabolism.48 Third, it has been suggested that bacterial isolates of gut microbiota may have pro- or anti-inflammatory properties, impacting weight as obesity, having been associated with a low-grade systemic inflammation corresponding to higher plasma endotoxin lipopolysaccharide concentrations defined as metabolic endotoxaemia.49, 50, 51, 52 Fourth, extracting crude fat in feed and excreta, Nahashon et al.53 reported that feeding laying Leghorn with Lactobacillus improved significantly retention of fat with increased cellularity of the Peyer's patches of the ileum, which indicated ileal immune response. Conversely, Bifidobacterium and Lactobacillus species have been cited to deconjugate bile acids, which may decrease fat absorption.54

Finally, specific strains of Lactobacillus and Bifidobacterium fed to farm animals have been shown to increase daily weight gain,55 and this fact has been used for decades in agriculture to increase feed conversion. In this context, one cannot exclude that the ‘growth promoter’ effect in animals associated with oral administration of specific probiotics strains is similar to the mechanisms involved in human obesity. For instance, Abdulrahim et al.56 reported that L. acidophilus significantly increased abdominal fat deposition in female chickens when administered alone and up to 31% when it was associated with zinc bacitracin. Further studies are therefore mandatory in exploring the interactions between probiotics and weight regulation.