The gut microbiome plays a crucial role in maintaining gastrointestinal homeostasis in humans, among others, by producing bioactive metabolites and limiting intestinal colonization by pathogenic microorganisms. Disruption of this balance, referred to as dysbiosis, leads to a range of adverse changes, including reduced microbial diversity, a relative increase in potentially pathogenic bacteria, and a concomitant decrease in beneficial commensal bacteria [17]. Although studies in humans are essential for translational analyses of the gut–brain axis, the majority of mechanistic knowledge derives from animal models, which allow controlled, precise manipulation of the gut microbiome and appropriate behavioral testing. Findings from studies using germ-free animals and fecal microbiota transplantation indicate that the composition of the gut microbiome significantly modulates stress responses and anxiety- and depression-like behaviors, thereby confirming its key role in regulating gut–brain axis functioning [17]. Experimental oral administration of the human-derived inflammatory E. coli K1 strain in mice induced colitis, increased blood LPS levels, hippocampal inflammation, cognitive decline, and depression-like behaviors, suggesting that inflammatory gut bacteria can influence intestinal and brain function [108]. The gut microbiota plays a key role in modulating central neurotransmission and brain function by influencing major neurotransmitter systems and metabolic pathways [109,110,111]. Mesolimbic circuits have been proposed as potential targets through which microbiome-related interventions may influence behavioral responses to drugs of abuse, including alcohol and opioids [17]. The concept of microbial endocrinology emphasizes that microorganisms inhabiting the gastrointestinal tract can produce and recognize the same neuroendocrine hormones as the host, thereby enabling bidirectional communication within the gut–brain axis [112]. This bidirectional communication is further supported by the identification of the two-component sensor kinase quorum-sensing E. coli regulator C (QseC) in E. coli, which has been shown to directly sense host-derived epinephrine and norepinephrine, undergo autophosphorylation, and transmit the signal to its cognate response regulator [112,113]. Biochemical analyses have demonstrated that QseC undergoes autophosphorylation upon exposure to epinephrine or norepinephrine and subsequently transfers the phosphate group to its response regulator quorum-sensing E. coli regulator B (QseB), thereby modulating transcriptional programs in E. coli [113].

Some gut bacteria, including E. coli, can produce neuroactive compounds such as norepinephrine and modulate tryptophan metabolism, which indirectly affects serotonergic neurotransmission. In this way, E. coli may participate in gut–brain axis signaling and indirectly influence CNS function [109]. It has been demonstrated that members of the gut microbiota can synthesize neurochemicals such as GABA, dopamine, norepinephrine, serotonin, acetylcholine, and histamine using biochemical pathways identical to those found in vertebrate tissues [112]. In E. coli, GABA is synthesized from glutamate by the pyridoxal-5′-phosphate-dependent enzyme glutamate decarboxylase (GadB), which forms part of the glutamate-dependent acid resistance system [114]. Capitani [114] showed that GadB activity is strongly pH-dependent and that the enzyme undergoes conformational rearrangements that regulate access to its active site. GadB is a hexameric enzyme and, together with the antiporter GadC, constitutes the glutamate-dependent acid resistance system that mediates proton consumption in E. coli. Exposure to cocaine may indirectly affect the composition of the gut microbiota by increasing intestinal norepinephrine levels, thereby promoting colonization by certain commensal bacteria, including E. coli [115]. Norepinephrine can modulate interactions between commensal and virulent E. coli strains, including O157:H7, and the intestinal epithelium, thereby promoting the early stages of bacterial adhesion to host cells [116]. The QseC-dependent signaling pathway regulates flagellar gene expression and contributes to virulence gene regulation in enterohemorrhagic E. coli, linking host catecholamine signaling with bacterial behavioral responses. Deletion of qseC attenuates virulence in a rabbit infection model, supporting the functional relevance of this signaling pathway in host–pathogen interaction [113].

Alterations in the composition of the gut microbiota may lead to modifications in the host metabolic profile, particularly to a reduction in glycine availability in the intestine, blood, and cerebrospinal fluid, which is associated with an exacerbation of behavioral responses, such as enhanced cocaine-induced locomotor activity. It has also been demonstrated that interference with microbiota-dependent glycine metabolism, including through glycine supplementation or the use of bacterial strains incapable of glycine uptake, reverses the observed behavioral changes, thereby confirming the role of the gut microbiota in modulating interactions between the gastrointestinal tract and the CNS in response to psychoactive substances [115]. Overall, the available evidence indicates that microbial metabolic activity may contribute to behavioral sensitivity to drugs of abuse rather than merely reflect secondary alterations [17].

In vitro studies using human cells complement data derived from animal models and indicate that specific gut bacteria may directly modulate the expression of genes associated with addiction. It has been shown that stimulation of the human intestinal epithelial cell line HT-29 with pathogenic E. coli strains (the intracellular strain E. coli UM146 and the extracellular strain E. coli UM147), particularly the intracellular strain, leads to significant changes in the expression of genes belonging to signaling pathways associated with cocaine and amphetamine addiction, including genes involved in cognitive functions (GNAS, PPP1R1B), stress responses (JUN, CREB1), and anxiety (BDNF). Concurrently, the observed downregulation of the FOSB gene, which participates in the organism’s response to morphine, suggests a potential association between bacterially induced changes in gene expression and mechanisms underlying opioid response [117]. Such alterations are consistent with evidence suggesting that immune and microbiota-related signaling pathways may influence brain function and reward-related processes implicated in addiction [11]. One mechanism potentially linking the gut microbiota, including Gram-negative bacteria such as E. coli, to the gut–brain axis and the pathophysiology of affective disorders and addictions is the activation of Toll-like receptors, particularly TLR4. Genetic studies in mice demonstrated that mutations in the Lps locus, identified as the Tlr4 gene, abolish cellular responsiveness to LPS, indicating that TLR4 is essential for LPS signal transduction. In C3H/HeJ mice, a missense mutation in Tlr4 results in substitution of proline with histidine at position 712 within the cytoplasmic domain of the receptor, whereas C57BL/10ScCr mice carry a null mutation associated with the absence of Tlr4 mRNA expression. These findings established that TLR4 is required for recognition of LPS and for downstream signaling in response to Gram-negative bacteria [118]. Activation of TLR4 in the CNS may lead to microglial activation and increased production of proinflammatory cytokines, thereby promoting the development of neuroinflammation, which has been associated in numerous studies with the occurrence of depressive and anxiety-related symptoms [119]. Consistent with this pathway, oral administration of the inflammatory E. coli K1 strain in mice has been shown to increase fecal and circulating LPS levels, activate NF-κB signaling in the colon and hippocampus, elevate tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) expression, and increase ionized calcium-binding adaptor molecule 1-positive microglial activation in the hippocampus [108].

Opioid use may lead to impaired intestinal function, including weakening of the intestinal barrier and bacterial translocation, as well as alterations in the profile of microbial metabolites [2,17]. Morphine treatment promotes the translocation of E. coli from the intestinal lumen to the mesenteric lymph nodes and the liver, which is accompanied by increased intestinal epithelial permeability resulting from impaired intestinal barrier integrity [23]. Subcutaneous implantation of slow-release morphine pellets is widely used as an experimental model of chronic opioid exposure in rodents. It produces measurable systemic morphine levels over several days with a characteristic pharmacokinetic profile [120]. Using this experimental approach, Meng et al. [23] demonstrated that morphine up-regulates TLR2 and TLR4 expression at both mRNA and protein levels in small intestinal epithelial cells and subsequently activates TLR-dependent signaling pathways that disrupt tight junctions between enterocytes. This disruption increases intestinal barrier permeability and facilitates bacterial translocation into the host tissues. The authors demonstrated it experimentally in wild-type mice by subcutaneously implanting a 75 mg morphine pellet or a placebo pellet. Then, after 24 h, they collected mesenteric lymph node and liver suspensions, which were cultured on blood agar plates overnight for quantitation of colony-forming units (CFUs). Homogenates obtained from morphine-implanted mice contained significantly higher numbers of CFUs in contrast to animals not treated with morphine. Moreover, they proved that in MOR knockout mice, this effect did not occur. However, most available evidence derives from acute, high-dose morphine paradigms, which may not fully recapitulate the fluctuating exposure patterns characteristic of chronic human addiction. Furthermore, the pharmacokinetic profile of pellet implantation differs from that of intermittent administration, which allows greater control over dosing schedules and produces distinct patterns of drug exposure.

Alterations in the gut microbiota composition are an essential factor in the pathogenesis of many diseases, among which IBD is particularly significant. Although the etiology of IBD is complex and multifactorial, dysbiosis is associated with increased levels of E. coli, Mycobacterium spp., and C. difficile, which are regarded as key microorganisms linked to the development of this disease [12]. Increased intestinal permeability resulting from epithelial barrier dysfunction has been reported in clinical studies in patients with IBD, IBS, alcoholic liver disease, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, liver cirrhosis, severe acute pancreatitis, primary biliary cholangitis, type 1 and type 2 diabetes, as well as depression [19]. In line with these observations, experimental studies in animal models indicate that morphine can alter gut barrier function and host–microbial interactions, promoting bacterial translocation and infection-related outcomes, as summarized in Table 1.