Impact of nanosilver on gut microbiota: a vulnerable link

Review For reprint orders, please contact: [email protected] Impact of nanosilver on gut microbiota: a vulnerable link Dinesh Kumar Dahiya*,‡,1 , Renuka‡,2 & Anil Kumar Puniya3 1 Advanced Milk Testing Research Laboratory, Post Graduate Institute of Veterinary Education & Research, Rajasthan University of Veterinary & Animal Sciences at Bikaner, Jaipur 302020, Rajasthan, India 2 Department of Veterinary Physiology & Biochemistry, Post Graduate Institute of Veterinary Education & Research, Rajasthan University of Veterinary & Animal Sciences at Bikaner, Jaipur 302020, Rajasthan, India 3 College of Dairy Science & Technology, Guru Angad Dev Veterinary & Animal Sciences University, Ludhiana 141004, Punjab, India * Author for correspondence: Tel.: +91 96 4948 3000; [email protected] ‡ Authors contributed equally A plethora of nanoparticles are currently used in the food industry in myriad applications. Of these, ‘nanosilver’ is widely used due to their multitude actions. Recent consensus among the scientific community affirmed that nanosilver might potentially alter the gut microbiota instead of their intended use that has a profound effect on our health. Dysbiosis of gut microbiota led to the onset of serious pathological conditions as reflected from several studies. In lieu of the positive impact of nanosilver, their inadvertent toxic effects on gut microbiota are underestimated. In this review, first all studies concerning the influence of nanosilver on gut microbiota are discussed along with relevant pharmacokinetic studies and in closing section the challenges and future task remained in the field are highlighted. First draft submitted: 9 June 2017; Accepted for publication: 24 October 2017; Published online: 21 December 2017 Keywords: dysbiosis • GI tract • gut microbiota • nanoparticle • nanosilver In the recent years, nanotechnology has emerged as a powerful scientific discipline as evidenced by its unprecedented spread in all spheres of life. Nanoparticles (NPs), the working weapons of this technology, are extremely smaller particles having a diameter of 100 nm or less [1]. Because of their preferred size and properties they are extensively utilized in various fields for different purposes. For example, in cosmetics they act as ultraviolet filters, in toothpaste they resist biofilm formation, in food-processing industries they aid in improving various physicochemical properties of the food like palatability, nutrient bioavailability and extended release of bioactive components [2,3]. Besides, NPs are an integral part of the food packaging industry as they not only increase the mechanical strength of the packaging material but also the shelf-life of the products [4,5]. Recently, NPs have also been employed to fabricate nanosensors to detect microbial and pesticide contamination in food [6,7]. Not only this, NPs have also become an excellent tool in the diagnosis and treatment of various life-threatening disease such as cancer because of their high surface-to-mass ratio, quantum properties and ability to adsorb and carry other compounds such as drugs, probes and proteins to their target site [8–10]. For effective results, NPs should cross the biological barriers to deliver therapeutic agents to the cells and tissues involved in pathogenesis [11,12]. Since the use of NPs either directly or indirectly has been increased tremendously in our daily life, therefore there are huge chances that they will intentionally or inadvertently affect the other physiological processes besides the targeted one. Thus, the matter is of great concern and fewer investigators are active in exploring the adhered consequences. Among major NPs viz TiO2 , copper (Cu), silver (Ag) and gold (Au) are used in food industry, Ag-NPs or ‘nanosilver’ are preferentially being used because of better antimicrobial activity. Ag-NPs are also the first choice for the formulation of other consumer products as evidenced from a market survey report in which it is documented that out of 846 NPs-based products advertised in market 435 products were formulated using Ag-NPs [13]. Most of the drugs/food products containing Ag-NPs are meant for oral consumption as it is the most economical, noninvasive and effortless route available for the delivery of these NPs. The estimated dietary intake of Ag-NPs by an individual is approximately 70–90 µg/day but the exact level would be quite high due to many fold increase in Ag-NPs use in the food industry [14]. Ag-NPs showed very limited toxicity on eukaryotic cells, but we cannot C 2017 Future Medicine Ltd 10.2217/fmb-2017-0103  Future Microbiol. (Epub ahead of print) ISSN 1746-0913 Review Dahiya, Renuka & Puniya deny their adverse side effects due to bioaccumulation in the body. However, only fewer in vivo studies are available and that too is inconclusive. On the other hand, Ag-NPs demonstrate strong antimicrobial attribute mediated through release of Ag ions [14–16]. Ag-NPs are bactericidal against Gram-negative and -positive bacterial strains, based on the thickness of peptidoglycan layer. Ag ions disturb the bacterial wall integrity via interacting with the negatively charged peptidoglycan [17]. These NPs can decimate bacteria entering through the cell wall, interfere with the respiratory chain and phosphate intake, which may further impair the DNA replication and protein modification [18]. The antibacterial attribute of Ag-NPs might disturb the gut microbiota and consequently the human health. Only a few studies have addressed the subject ‘nanosilver–gut microbiota’. Therefore, in this review, efforts are made first to discuss the studies in context to Ag-NPs and gut microbiota modulation along with their relevant pharmacokinetics studies. In the later half, the conclusion drawn from the elaborated findings and what are the future task remained in the field will be discussed. The discussion of all other NPs is beyond the scope of this review. Before proceeding to the main topic, a brief introduction about gut microbiota and its relevance in human health is also discussed for better interpretation. Gut microbiota: an overview GI tract despite of its pivotal role in digestion and uptake of nutrients, immune modulation also harbors trillions of microbes that actively influence the host physiology. Gut-situated diverse communities of microbes contain greater than 1000 different bacterial species that altogether represent 1014 –1015 microbes [18–20]. The population of microbes present in the GI tract is ten-times the total number of cells a eukaryote has and help in carrying out the metabolic activities that are associated with host physiology. GI tract is almost sterile at the birth and bacterial communities start colonizing during delivery. During the lifespan, there is a dramatic shift in microbial populations because of several inherent and external factors. However, it becomes almost consistent during the adolescence where few microbial communities showed the predominance. A typical gut microbiota is the signature profile of a particular host. In fact, population can be identified/differentiated on the basis of microbial signatures or ‘fingerprints’ only. Majority of the colonized bacterial species in an adult human being fall in the four major phyla, Gram-negative Bacteriodetes, Gram-positive Firmicutes (together constitute 90% of the total bacterial population), Proteobacteria and Actinobacteria [21,22]. Before the inception of the 21st century, it has remained a neglected organ but lately it is elucidated that it has a crucial role in maintaining the well-being. However, switch in microbial homeostasis called as ‘dysbiosis’ is predicted as one of the key factors in the progression of several diseases such as cancer, obesity, Crohn’s disease, Type II diabetes, celiac disease and neurobehavioral alterations [23–25]. Investigators have used different strategies to elucidate the effect of diet, probiotics, prebiotics in maintaining the gut health and pathological conditions [26,27]. Probiotics have the ability to refurbish the altered gut microbiota by adhering to the gut epithelial cells [28] and stimulating the growth of beneficial microbes. Not only this, probiotics simultaneously helps in improving the physiological condition by the production of bioactive compounds [29,30]. However, investigations concerning the effect of Ag-NPs on gut commensal bacteria and their probable consequences on the host are in the initial stages. These NPs hold potential to profoundly disrupt the gut homeostasis and in turn the host physiology. The effect of external agents on gut microbiota is commonly ascribed by means of two different analysis, in other words, α-diversity (depicts overall diversity in a sample) and β-diversity (compare the differences in bacterial population between the different samples). In the forthcoming section, we have ascribed all the available studies that concerning the impact of Ag-NP on the gut microbiota. Implication of nanosilver on gut microbiota Evidence from rodent & human studies To devise the effect of Ag-NPs on gut microbiota is a burgeoning topic worldwide. The first study which showed Ag-NPs feeding have gut microbiota modulation affects in rodents comes from Williams et al. [15]. They evaluated that oral feeding of Ag-NP in Sprague–Dawley rats for 13 weeks resulted in a size- and dose-dependent change in ileum microbial population and gene expression. The results depicted an increase in the size and shift of gut microbial populations toward greater proportions of pathogenic Gram-negative bacteria and decrease in proportion of Firmicutes and Bacteroidetes. They portrayed that the change of Firmicutes/Bacteroidetes ratio was due to the reduction in Lactobacillus and Bifidobacterium populations. Further analysis of rat ileal tissue genes involved in immune modulation viz MUC3, TLR2, TLR4, GPR43 and FOXP3 revealed that lower concentration of Ag-NP also has the potential to suppress their expression. The dynamics of Foxp3 and TLR2 reported in this study might be due to the immune tolerance mediated by regulatory T cells [31]. These findings forced the authors to conclude that 10.2217/fmb-2017-0103 Future Microbiol. (Epub ahead of print) future science group Impact of nanosilver on gut microbiota: a vulnerable link Review oral exposure of Ag-NP has the ability to disrupt the intestinal microbiota that in turn modulated the gut-mediated immune response. However, the exact mechanism by which the Ag-NP modulates the intestinal immune response is yet to be elucidated. But it was proposed that extended exposure of Ag-NPs to gut microbiota favors their binding to Gram-positive bacteria instead of Gram-negative bacteria due to the presence of predominant charge group on their cell wall. This increases the decimation of Gram-positive bacteria in the gut and thereby, provides opportunity for Gram-negative bacteria to interact more with the epithelial molecules (TLR 2 and 4, NOD2 – an intracellular pattern recognition receptor), MUC2 and MUC3 glycoproteins secreted from goblet cell in large intestinal epithelial lining and ultimately modulate the immune system [15]. Besides that the immune cells present in intestinal mucosa such as dendritic cells or macrophages might uptake these Ag-NPs inside the epithelial lumen. Such engulfment might stimulate the host immune cells to synthesize and secrete inflammatory cytokines. In a similar but different study, the investigators demonstrated the effect of varying concentration of Ag-NP for 28 days in mice [32]. The outcomes showed a significant decrease in bacterial richness at the phylum level. A dose- and size-dependent decrease in Firmicutes and increase in Bacteroidetes was observed for Ag-NP. In a recent study, the authors tested the impact of varying dosages of dietary Ag-NP on the gut microbiota in mice during 28 days of experimentation. The concentrations of Ag-NP adopted in the study were nearly similar that an individual encounters normally via daily food intake. Body weight and physiological parameters’ results revealed no overt toxicity in animals. However, Ag-NP feeding altered α-diversity (bacterial evenness) as well as β-diversity (population) indexes in a dose-dependent manner. Ag-NP feeding also elicited the Firmicutes/Bacteroidetes ratio which was primarily due to increasing in Lachnospiraceae and S24–7 family. These shifts observed in response to Ag-NPs treatment were similar to those found in pathological conditions such as obesity and diabetes. Also, it is quite feasible that the same consequences will be obtained with human gut microbiota if they would be exposed to similar Ag-NPs concentration and potentially instigate the gut inflammation associated with obesity and other linked diseases [17]. These findings were further supported by subsequent studies from a group in which Ag-NP feeding not only altered the gut microbiota but also elicit the inflammation in the intestine [33]. Interestingly, the gut dysbiosis effects were still persistent when the amount of Ag-NP (20 mg/kg per dose) was restricted below the recommended dosage for human ingestion [34]. More specifically, Ag-NP shifted the Firmicutes/Bacteriodetes ratio by reducing the subpopulation of the genus Lactobacillus. The shift was understood as a probable cause of inflammation similar to as noticed in other inflammatory disorders as disclosed above. The hypothesis that Ag-NPs interactions with gut microbiota have some consequences on changes on neurobehavioral activity was addressed very recently. Supplementation of Ag-NP (3.6 mg/kg body weight [bw]) of two different shapes, in other words, cube and spheres for short duration in Sprague–Dawley rats have different gut alterations affect. Ag-NP in cube form led to a reduction in Clostridium spp., Bacteroides uniformis, Christensenellaceae and Coprococcus eutactus populations, whereas Ag-NP in spherical form decreased Oscillospira spp., Dehalobacterium spp., Peptococcaeceae, Corynebacterium spp., Aggregatibacter pneumotropica populations. The authors correlated these gut microbial shifts with the behavioral changes evaluated by the elevate plus maze test specifically perform to examine anxiety and exploratory activities in animals [25]. In contrast, Hadrup et al. noticed nonsignificant alterations in the Firmicutes and Bacteroidetes population with no toxicological effects on animal’s physiology during 28-day Ag-NPs feeding trial [35]. Likewise, another study also reported no considerable modulations in the gut microbiota of mice after exposure with Ag-NPs of different sizes and concentrations [16]. Although human in vivo studies are missing and only one study has evaluated the effect of Ag-NPs on the imprints of human fecal material, under in vitro conditions. The study assessed the microbial shift in a synthetic culture designated as microbial ecosystem therapeutic-1 established from the stool of healthy donor. After encountering the consortium with various concentration of Ag-NP (0–200 mg/l) for 48 h, a negative shift in the microbial community toward pathogenic bacteria was observed as revealed by analyzing gas production, fatty acid methyl ester profile, PCR-denaturing gradient gel electrophoresis profiles and 454 pyrosequencing data. Ag-NP treatment decreased Bactroides ovatus by 57%, whereas Raoultella sp. and Escherichia coli proportions were elicited by 28–46% and 50–80%, respectively. Not only this, a reduction in proportions of other bacterial species like Roseburia faecalis, Rosuburia intestinalis, Eubacterium rectale and Ruminococcus torques were also observed when compared with control. The authors portrayed that Ag-NP ingestion either intentionally or inadvertently could have a deleterious effect on intestinal microbiota [36]. Herein, we noticed discrepancies between the rodent studies in term of gut microbiota alterations and the probable reason for the same might be due to the variation in size, concentration, composition, aggregation state of the Ag-NPs, duration of treatment, mode of administration and composition of the feed offered to the animals. In future science group 10.2217/fmb-2017-0103 Review Dahiya, Renuka & Puniya addition, the methods and analysis technique used for hunting and interpretation of the gut microbiota dysbiosis also played a significant role. As some investigators have used global sequencing method [16,25] for analysis while others have opted real-time PCR [15,35] or next-generation sequencing as a method of choice [17]. The species–species interaction and the methods used for the microbial analysis might also account for these inconsistencies in the outcomes. Evidence from other species The effect of Ag-NPs in context to gut microbiota has also been studied in nonrodent studies. Sawosz et al., in their investigation on quails (Coturnix coturnix japonica, 10-day old), observed that Ag-NP only in high concentration, in other words, at 25 mg/kg has profoundly increased the number of Gram-positive bacteria such as Lactobacillus spp, Leuconostoc lactis, Actinomyces naeslundii as compared with their control counterparts. A slight but nonsignificant increase in the number of Streptococcus bovis was also observed (Table 1), while no change in Enterococcus faecium, E. coli and other members of Enterobacteriaceae was revealed [37]. One limitation of this study was that the authors used conventional media-plating technique for microbes’ assessment. Similar results were obtained from a different research group on gut microbiota of weaned pigs in terms of coliforms while contrasting outcomes for Lactobacillus populations when analyzed using fluorescent in situ hybridization [38]. Merrifield et al. in zebrafish (Danio rerio) model elucidated that Ag-NP (500 mg/kg food) meshed feed offered for 14 days caused changes in the richness and diversity of gut microbiota [39]. However, no difference was visualized during the histological examination of the intestinal epithelium. Exposure of tilapia (Oreochromis niloticus L.) with sublethal dosages of Ag-NPs (0.8 and 0.4 mg/l) for 3 weeks caused a substantial decrease in intestinal wall thickness and inflammation of mucosal layer. In addition, elevated catalase expression was also observed in Ag-NP-treated group. Gut microbiota was also depleted post-Ag-NP treatment in a dose-dependent manner with a subsequent upregulation in glutamate dehydrogenase activity [40]. Exposure of Drosophila melanogaster with Ag-NPs induced a prominent reduction in gut microbiota diversity as determined by 16S rRNA pyrosequencing. It strikingly elicited Lactobacillus brevis population whereas, resulted in alleviation of Acetobacter proportion. It was noticed that Ag-NPs treatment retarded the developmental success but did not affect the longevity and reproduction. However, the study was not able to devise a definite conclusion whether the toxic effects of Ag-NP were either due to interaction with larval cells or with its intestinal microflora [41]. From the above studies carried out in different species, it is difficult to draw a correlation between Ag-NP treatment and alterations in gut microbiota. As all the studies have used different models for the evaluation of gut microbial dysbiosis and therefore, it is difficult to make a cross-study comparison. In spite of that, some of the above-mentioned studies reported similar changes in gut microbiota with respect to Lactobacillus population. Nevertheless, one advantage of assessing Ag-NPs or other NPs across different model species helps in concluding that the effects are not restricted to one taxa but simultaneously aid in elucidating the microbial species, most influenced by the same. Pharmacokinetics & distribution of nanosilver As described above, the use of the Ag-NP has been increased tremendously over the last decade and hence it is utmost important to know their pharmacokinetic properties for probable risk factors and safe use. Pharmacokinetics deals with assessing the absorption rate, how they have distributed and the way they are metabolized and excreted from the system using experimental models and mathematical approaches [42]. In spite of a plethora of papers on the medical use and biodistribution, exhaustive studies on the pharmacokinetics of NPs including Ag-NPs are scanty. Moreover, only a few studies were focused on physiological-based pharmacokinetic modeling (PBPK). PBPK is a comprehensive analysis about the NPs from absorption till its elimination using mathematical modeling. The likely benefit of PBPK is to determine the dosimetry at target tissue sites for nanomedicine applications. These gaps in the knowledge put several question marks in this area and might discourage their future use in the food sector and clinical applications. In the next section, we briefly described how the dissolution of orally ingested Ag-NP takes place in the gut environment. Chemical transformation of Ag-NPs in gut conditions Soon after their ingestion via different means, Ag-NP reaches the stomach and encounters with the present low acidic conditions (∼pH 1.5). The interaction led to the dissolution of approximately 97% Ag-NP into Ag+ ions within 5–6-h period as revealed in simulated gastric conditions. Furthermore, changes in the pH and ionic strength 10.2217/fmb-2017-0103 Future Microbiol. (Epub ahead of print) future science group future science group Table 1. Compilation of different experimental studies elucidating the effect of silver nanoparticles on the gut microbiota. Size and dosage Animal model Methods Influence on gut microbiota Ref. Ag-NPs 0, 5, 15, 25 mg/kg for 12 days Quail (Coturnix coturnix japonica) Colony count ↑ Lactobacillus spp, Leuconostoc lactis, Actinomyces naeslundii, Streptococcus bovis, NC in E. coli and Enterobacteriaceae [37] Ag-NPs Group 1 (0, 25, 50 and 100 µ g Ag/g), Group 2 (0, 20 or 40 mg Ag/kg) Piglets FISH ↓ Bifidobacterium, ↑ C. coccodes/Eubacterium, NC in coliform and Lactobacilli [38] Ag-NPs 3% body weight/day for 14 days Zebra fish (Danio rerio) PCR and DGGE No significant changes in gut microbiota [39] Ag-NPs 0.8 and 0.4 mg/l for 3 weeks Tilapia (Oreochromis niloticus L.) SDS-PAGE Significant change in the gut microbiota [40] Ag-NPs 2.25, 4.5 and 9 mg/kg bw, 28-day oral dosing Wistar Hannover Gales rats qPCR NC in Firmicutes, Bacteroidetes [35] Ag-NPs 25, 100, 200 mg/l of Ag-NP and 2.5, 10, 20 mg/l PVP suspension for 48 h Synthetic stool cultute MET-1 cultures PCR-DGGE, 454 Pyrotag sequencing, bacterial gas production, fatty acid analysis ↓ Bactroides ovatus, Roseburia faecalis, Rosuburia intestinalis, Eubacterium rectale and Ruminococcus torques, ↑ Raoultella sp, E. coli [36] Ag-NP (7 µ m and 450 µ g/ml) Drosophila melanogaster PCR ↓ Acetobacter ↑ Lactobacilli [41] Citrate-stabilized Ag-NP (10, 75 and 110 nm), (9, 18 and 36 mg/kg bw per day) for Sprague–Dawley rats 13 weeks PCR ↓ Firmicutes, Lactobacillus ↑ Bacteroidetes, Bacteroides, Bifidobacterium, Enterobacteria [15] Ag-NP (0, 46, 460, 4600 ppb) for 8 months C57BL/6 female mice PCR, NGS ↑ Firmicutes/Bacteroidetes ratios, ↓ Lactobacillaceae, Bacteroidaceae [17] PVP or citrate Ag-NP (20, 110 nm), (10 mg/kg bw/day), 28 days and oral dosing C57BL/6NCrl mice PCR No significant change in gut microbiota [16] Ag-NP 2.5 mg/kg bw per day for 7 days Male CD-1 mice 16S rRNA profiling by 454 pyrosequencing ↓ Firmicutes/Bacteroidetes ratios, ↓ Lactobacilli [34] Ag-NP 3.6 mg/kg bw of cube and spherical shape Sprague–Dawley rats 16s rRNA sequencing ↓ Clostridium spp, Bacteroides uniformis, Christensenellaceae and Coprococcus eutactus (cube), ↓ Oscillospira spp, Dehalobacterium spp, Peptococcaeceae, Corynebacterium spp, Aggregatibacter pneumotropica (spheres) [25] Ag-NP: Silver nanoparticle; bw: Body weight; DGGE: Denaturing gradient gel electrophoresis; FISH: Fluorescent in situ hybridization; MET: Microbial ecosystem therapeutic; NC: Non significant; NGS: Next-generation sequencing; PVP: Polyvinylpyrrolidone. Review 10.2217/fmb-2017-0103 Impact of nanosilver on gut microbiota: a vulnerable link Nanoparticles Review Dahiya, Renuka & Puniya during their transit through gut critically affect the properties of the Ag-NPs. The food constituents, mucus, digestive enzymes and bile salts exude in the gut have a profound effect in altering the properties of the Ag-NPs in the GI tract [43,44]. The concentration of free Ag+ ions in the gastric fluid is very low (10-9 M) because of its precipitation with Cl- ions. The maximum amount to which soluble silver species (sum of Ag+ , AgCl(aq), AgCl2 and AgCl3 2- ) exists in the synthetic gastric fluid was estimated as 0.51 mg/l. Beyond this limit, AgCl(s) will form and restrict the extra increase in Ag bioavailablity. Absorption of Ag-NP from the GI tract The in vivo studies to understand the uptake of Ag-NPs on the intestinal cells and on their permeability are still missing. Only studies available are with the cell line models because it is comparatively easier to manipulate the cell lines as per the experiment and for a better conclusion of the results. Intestinal epithelial lining and mucus are the main barriers that interferes with the permeability of Ag-NPs in the lumen. Most in vitro studies in context with Ag-NPs were carried on Caco-2, HT-29 and T84 epithelial cells and all studies emphasized that the transportation through the gut barrier is dependent upon the size and dosage of NPs [45–50]. Smaller Ag-NPs were found to cross the barrier more readily than the larger ones and found to alter the permeability of intestinal epithelium that might have deleterious consequences on the host health. Ag-NPs uptake in the epithelial cells was possible either through clathrin-dependent endocytosis or via M cells, however, still more clear research is required to understand the translocation process. We believe that studying the Ag-NPs interaction on ‘organ-on-a-chip model’ that closely mimics the in vivo gut environment substantially improve our understanding about the uptake and translocation mechanisms. Biodistribution of Ag-NP in systemic organs To date, the majority of the studies focused on the distribution of Ag-NP in the systemic organs were studied by administration of either a subcutaneous or intravenous dose and their discussion is beyond the scope of this review. Only studies performed with oral exposure of Ag-NP are described here. Park et al. reveled that when 1 mg/kg or 10 mg/kg citrate-coated Ag-NP was fed to rats for 96 h, the bioavailability in the blood of animals was 1.2 and 4.2%, respectively. This signifies that the bioaccumulation of the NPs is directly dependent on the dosage [51]. In another study, the authors found that Ag particles were accumulated in the higher amount in liver and spleen instead of other tissues evaluated. Moreover, the amounts of Ag found in AgNO3 (9 mg/kg bw)-treated rats were comparably higher than Ag-NP (90 mg/kg bw)-treated group [52]. These findings were in concordance with another study depicted the similar pattern of Ag bioaccumulation from ionic silver source (silver acetate 9 mg/kg) than Ag-NPs (12.6 mg/kg) [53]. Therefore, there is still a lot of scope available for further research in this direction to come out with a precise conclusion on the aforementioned subject. Conclusion & future perspective In the last couple of years, the application of nanotechnology, especially the Ag-NP, has tremendously increased in the food industry due to several advantages. However, no considerable attention has been sought from food regulatory authorities on the probable consequences that may be raised due to their toxicological effect on the host physiology via oral exposure and is a matter of concern. Intestinal microbiota which is now wholly viewed as a separate vital organ has been elucidated to play a pivotal role in maintaining the positive health status by influencing various mechanisms. It is speculated as the major primary site where Ag-NP ingested via food or other means actually interacts with gut microenvironment and implicates the microbial communities. Moreover, it is believed that mucus present in the gut lining hinders the Ag-NPs absorption into the intestinal epithelial cells and that increases their interaction with gut microbial communities. The studies summarized here undoubtedly evidenced that Ag-NPs have the potential to alter the gut microbiota and might make the host susceptible to certain diseases. A hypothetical figure that illustrates the interaction of Ag-NP with gut microbiota and probable implication on the host is depicted in Figure 1. As this switch in microflora documented in some of the studies is similar to those observed in pathological conditions. Although a clear correlation has yet to be established and requires further research. As several factors like size of Ag-NP, animal model opted for study and type of analysis performed impart ambiguity between the studies that hindered in getting a clear consensus. Also, most of the studies were conducted in rodents and none in the human volunteers and there is a vast difference between the gut microbiome of these two species. So, we cannot correlate the results obtained in rodents with human gut microbiota. The sampling site is also a matter of concern to reflect the changes; as microbial diversity varied across 10.2217/fmb-2017-0103 Future Microbiol. (Epub ahead of print) future science group Impact of nanosilver on gut microbiota: a vulnerable link Review Alteration of gut microbiome Ingestion of Ag-NP Gram-positive bacteria Ag-NP Gram-negative bacteria Goblet cells Goblet cells T cells DCs MΦ Epithelial cells T cells Modulation of intestinal immune response Pathogenesis of several diseases Type 2 diabetes Celiac disease Cancer Obesity Figure 1. Schematic representation of interaction of nanosilver with the gut microbiota and the pathological consequences. (A) Represents intact Ag-NPs. (B) Alteration of the gut microbiota (both Gram-positive and -negative bacteria). (C) Modulation of intestinal immune response. (D) Altered microbiota involvement in the pathogenesis of several diseases, in other words, cancer, obesity, Type II diabetes, celiac diseases. Ag-NP: Silver nanoparticle; DCs: Dendritic cells; M: Macrophage. the gut with more differences in the large intestine in comparison to the small intestine. However, we cannot deny from the assumption that a recent rise in some of the inflammatory disease linked to gut microbiota might be due to the consequential inadvertent effect of Ag-NP on the gut commensal. From pharmacokinetics studies, it is inferred that the estimated bioavailability of Ag-NPs in the animal’s blood postoral absorption was 1–4.2% and liver and spleen were characterized as the principal organs for its deposition. However, the in vivo pharmacokinetics studies regarding Ag-NPs absorption through intestine are still awaited. Future in-depth studies are warranted in experimental disease models to derive a clear-cut picture concerning that the Ag-NPs influence the gut microbiota, especially the mechanistic insight. The physiological parameters like blood profile, histological analysis, impact on gut epithelial barrier, villi structure, mucus and gene profile should be correlated with dysbiosis. A universal approach should be followed in analyzing the gut microbial data generated from multiple species after treatment and should be deposited to a database that can be readily utilized by another research group for their reference as suggested by other [54]. Studies emphasizing on the toxicological effect of Ag-NP on ‘microbial signatures’ linked with beneficial traits such as Akkermansia muciniphila, Faecalibacterium prausnitzii, Bacteroides fragilis, B. uniformis, Eubacterium hallii and Clostridium cluster IV and XIVA members should be delineated. Fecal transplantation of gut microbiota from animals treated with Ag-NP to germ-free mice will definitely provide a better-dissected view of the matter. Not only this, the effect should also be addressed in relation to other microbial communities like viruses that are also the primary but neglected component of the gut [55]. Whether the gut microbiota would recover after the withdrawal of Ag-NPs from diet or the changes are permanent is still to be elucidated. Promising studies are focusing on the fact that which size and dosage would have a maximum impact on the gut microbiota are immediately warranted. With the advent of ‘omics’-based technology, it is quite feasible to understand the link not only at genomic level but also at transcriptomics and proteomics level that gives useful insights by correlating the interlinked mechanisms. Metabolomic analysis should also be performed post-Ag-NP exposure to find out the changes in metabolites profile future science group 10.2217/fmb-2017-0103 Review Dahiya, Renuka & Puniya in context with altered gut microbiome. In a nut shell, we believe that there is an emanate need from the scientific community to look in the issue. Executive summary r A plethora of nanoparticles (NPs) are used in food industry for a myriad of things. r A recent report advocates that ‘nanosilver’ is predominantly used among the various NPs for multitude action. r Recent findings had clearly indicated that dysbiosis in gut microbiota led to pathogenesis of certain diseases. r Silver nanoparticle (Ag-NP) instead of their targeted action also intentionally or inadvertently influences the gut microbiota, which is now viewed as a ‘vital organ’. r Some of the discussed in vivo and ex vivo studies suggest that Ag-NPs have the potential to modify the gut microbiota. r But the matter is still inconclusive as several factors like size, dosage, experimental model, duration of the study and technique used to assess gut microbiota impart ambiguity in the findings. r In-depth pharmacokinetics studies about the absorption of Ag-NP in humans are missing and only in vitro evidence are available. r In vivo studies in rodents suggest that the bioavailability of Ag-NP in blood was approximately 1–4.2% of the ingested dosage, and liver and spleen were found as the primary site for their deposition. r We advocate that oral ingestion of Ag-NP may disrupt the gut microbiota that may lead to several pathological conditions as the reported change in the gut microbiota in some studies is similar to that observed in obesity. r However, the correlation between disrupted gut microbiota in influence to Ag-NP and disease conditions has not been conducted yet. r It might be a possible reason for recent rise in lifestyle-related diseases in the modern world as diet may have severe consequences on the gut microbiota. r Future studies are warranted to establish a link between the modified gut microbiota with host physiology. In this matter, the experimental outcomes from germ-free models may be a ‘milestone’. r Promising studies involving meta-transcriptomics and meta-proteomics analysis of gut microbiota would better illustrate functional information on ‘nanosilver–gut microbiota link’ and help in materializing the issue. Acknowledgements The authors acknowledge R Gupta for seriously evaluating the manuscript. Financial & competing interests disclosure Renuka would like to acknowledge Council of Scientific and Industrial Research, Govt of India, for funding in the form of a CSIRSRF (09/135(0645)/2011-EMR-I). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. 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