**1. Introduction**

About a third of the population in developed countries is obese in some degree [1]. The WHO has proposed the classification of normal weight to be when the body mass index (BMI) ranges between 18.5 and 24.9 kg/m2, overweight (BMI 25–29.9 kg/m2), class I obesity (BMI 30–34.9 kg/m2), class II obesity (BMI 35–39.9 kg/m2), and class III obesity (BMI ≥ 40 kg/m2) [2]. Obesity itself is a health risk factor that influences the development and progression of various metabolic and cardiovascular diseases, such as dyslipidemia, type−2 diabetes mellitus (T2D), hypertension, and ischemic heart disease, thereby worsening the quality of life of patients and survival [3]. This non-communicable disease is also associated with low-grade, chronic systemic inflammation by dysregulation of adipokines and pro-inflammatory mediators (i.e., cytokines, chemokine), and subsequent alterations in the immune cell composition and distribution [4]. Obesity is a multifactorial disease and thus, therapeutic approaches should be diverse [5]. In this sense, dietary treatments associated with body exercise are primary anti-obesity approaches. Other strategies, such as behavior therapy, have been also considered [6]. However, long-term strategies with hypocaloric diets and physical exercise are not frequently attained, and psychological comorbidities may be chronic in these patients. In consequence, alternative procedures should be explored. In this regard, by percutaneous electrical neurostimulation (PENS) of the sensory nerve terminals located in dermatome T6, the gastric wall can be stimulated to produce distention in the fasting state, and to block contractions in the postprandial phase [7]. As a result, stomach emptying is slowed and early satiety is promoted. Moreover, the associated modulation of neuronal activities influences on appetite reduction [8]. Indeed, we previously demonstrated that PENS achieved a significantly greater weight loss than an isolated hypocaloric diet in patients with BMI > 30 kg/m2, and this e ffect could be due, at least in part, to ghrelin inhibition [9,10].

Importantly, pathogenesis of obesity has been linked with alterations in gu<sup>t</sup> microorganisms. The intestinal microbiota is composed of tens of trillions of microorganisms, including at least 1000 di fferent species of known bacteria, placed in the gu<sup>t</sup> lumen or adhered to the mucus layer [11]. The five dominant bacterial phyla in the human gu<sup>t</sup> are Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and Verrumicrobia [12]. The immunomodulatory bacteria are of grea<sup>t</sup> importance for local and systemic immunity, whereas the muconutritive microbiota are responsible of mucus layer formation, and the proteolytic bacteria have key metabolic functions in protein digestion [13]. Among other functions, microbiota participates in the metabolism of proteins, plant polyphenols, bile acids, and vitamins, and in the assimilation of non-absorbable carbohydrates by conversion into monosaccharides and short chain fatty acids (SCFA) and gases. However, though the intestinal microbiota is highly diverse in "healthy" individuals, those exhibiting adiposity, insulin resistance and/or dyslipidemia are characterized by low bacterial diversity [14]. Furthermore, obesity is associated with substantial changes in the composition and metabolic functions of bacteria, making an "obese microbiota", which involves a greater extraction of nutrients from the diet [15]. Bacteroidetes prevalence is generally lower in obese people, in contrast with that of Firmicutes. However, the complexity of how the gu<sup>t</sup> microbiome modulates obesity can be more than a simple disproportion between these commensal phyla [16,17]. In this line, di fferent probiotics have demonstrated that they can balance microbiota bacteria and subsequently reduce body weight and metabolic and cardiovascular factors [18]. Thus, the aim of this study was to investigate the e ffect of probiotics on anti-obesity actions of PENS in conjunction with a hypocaloric diet.
