*4.7. Antibiotics*

Although the development of antibiotics has made it possible to treat life-threatening infections, the use of antibiotics reduces the microbiota diversity in the GI-tract [197]. Consequently, the use of antibiotics may elevate the number of pathogenic bacteria, such as *Enterobacter, Klebsiella, Citrobacter, and Pseudomonas* and decrease anaerobic bacteria [197]. For example, a human study analyzing the short term parenteral-neonatal antibiotic usage showed that it reduced the number of protective *Bifidobacteria* in the first couple of months of life [198]. Supporting these results, Penders et al. not only found a decrease of *Bifidobacteria*, but also of *Bacteroides* when infants administered antibiotics [122].

Results concerning the correlation of early antibiotic use and later risk of developing ADHD seem to be incoherent. A Danish population-based cohort study did not find an association in sibling-stratified Cox model between antibiotic use in the first two years of life and the risk of developing ADHD [130]. Another study, however, using 871 European newborns examined the effects of early antibiotic treatment on cognitive functions with the help of IQ and reading tests, and on symptoms of ADHD using the mentioned Conners Rating Scale-Revised (CRS-R). Thereby they discovered that children who consumed antibiotics in the first year of life showed a reduced reading ability score, higher scores on the CRS-R, rated by parents, and increased symptoms of ADHD at the ages of 7–11 years. Nonetheless, this association was not made for babies and children that used antibiotics between the ages of 12 months and 3.5 years. This indicates that one of the vital factors for developing ADHD is the age in which the newborn consumes the antibiotics. It seems that during the first 12 months of life, important developments of the gut-brain axis take place, which when disrupted influence the neurodevelopment, and thus, the CNS in the long run [12]. These data, however, must be interpreted with caution, as this was not an RCT. Accordingly, direct causation between the antibiotic use and later seen ADHD cannot be correctly made [199].

#### *4.8. Probiotics*

By definition of the FAO/WHO probiotics are "live microorganisms which when administered in adequate amounts confer a health benefit on the host" [200]. Benefits of probiotics include reinforcing a more desirable environment in the gut, a healthy digestive system, and finally an adequate immune system [201]. Thereby probiotics help to sustain and produce healthy enzymes while eradicating potentially harmful pathogens [202,203]. Naturally occurring probiotic sources include lactic acid fermented vegetables, such as kimchi or fermented dairy products as, for example, yogurt [204].

The influence of probiotic strains on psychiatric diseases has been examined by multiple studies, concluding a positive effect on such illnesses and are, thus, described as "psychobiotics" [90]. An animal study using mice showed that probiotic ingestion of *Bifidobacterium longum* and *breve* led to a reduction of depression and anxiety symptoms [205].

The seminal study by Pärtty et al. researched the effects of probiotic use on the development of ADHD in children by randomly administering strains of *Lactobacillus rhamnosus* into 75 infants. The infants were monitored at three weeks, 3, 6, 12, 18 and 24 months, and finally, at 13 years of age. The authors concluded that at the age of 13 years, ADHD was diagnosed in 6/35 (17.1%) children using the placebo, whereas no children had this disorder in the probiotic group. These results, even if encouraging, do not identify any specific composition of the microbiota to the neurodevelopmental disease, and thus, might mean that probiotics decrease ADHD in a different way rather than influencing the composition of the microbiome [142]. However, it is important to conclude that these findings potentially represent a method to reduce the risk of developing ADHD.

#### **5. Discussion**

This literature review demonstrates that the ADHD population has a different gut microbial composition in comparison to healthy controls as the phylum Actinobacteria is more and Firmicutes less abundant in ADHD patients. The genus *Bifidobacterium*, belonging to the phylum Actinobacteria, seems to play a significant role in the pathogenesis of ADHD and is recurrently influenced by several factors. *Bifidobacteria* do not only protect the barrier function in the gut and support a healthy immune response [168], but also influence the dopamine system by elevating the production of CDT which increases phenylalanine levels, and finally, results in higher levels of dopamine. This review showed that *Bifidobacterium* was decreased in offspring that were born (i) via c-section delivery ([122,123], (ii) as preterms [144,206], (iii) were breastfed [164] or (iv) were given antibiotics in the first months of life [122,198]. All of these factors are simultaneously associated with an increased risk of developing ADHD. Nevertheless, using *Bifidobacterium* as a potential biomarker for diagnosis of ADHD seems uncertain due to varying results regarding *Bifidobacterium* levels in ADHD patients. Although Pärtty et al. observed decreased levels of *Bifidobacterium* in 3 and 6-month-old ADHD patients [142], Aarts et al. detected slightly increased levels of the genus using a larger sample size and a more sensitive methodology [69]. Thus, for future research, well-designed studies, using a larger sample size, are needed to deduce a definite correlation between levels of *Bifidobacterium* and ADHD and the importance of this genus as a biomarker.

Additionally, this article concludes that the concertation of neuroprotective BDNF, indirectly influenced by the microbiome [173], plays a vital role in the pathogenesis of ADHD. The majority of reports showed a negative correlation between levels of BDNF and ADHD [173,174,207]. As the levels of SCFAs [173], and PUFAs [185] are positively correlated with BDNF, omega-3 fatty acids may prove to be of therapeutic importance. So far, various studies have shown that adding PUFAs to the diet only marginally decreases the symptoms of ADHD [193,194,208]. Future studies could assess the effects of various concentrations of PUFAs and age at which these were ingested on the symptom development of ADHD. BDNF shows properties important for neurogenesis in the critical stages of neurodevelopment. The production of SCFAs by the microbiome has been positively associated with levels of BDNF [173]. Therefore, increasing SCFAs through fiber-rich nutrition in combination with the appropriate gut microbial composition could also be a beneficial means for the treatment of ADHD symptoms.

It is widely known that c-section delivery causes the offspring s microbiome to be more similar to the mother s skin rather than her vaginal flora. However, it is still under debate to what extent this change impacts the development of ADHD. We decided to concentrate on the more recent papers, that used a large sample size and a precise methodology by differentiating between elective and emergency c-sections. These studies show that not every c-section increases the risk of developing ADHD, but only those that were done intrapartum [129,130]. Although this correlation is most probably not due to a differing microbial composition and rather due to various confounders, such as gestational age and birth weight [130], it is still important to note that emergency c-sections bear an intrinsic risk for the offspring developing ADHD.

Additionally, it has become increasingly clear to what extent prematurity plays a role in the development of ADHD. As the GI-tract and its colonization with bacteria are still underdeveloped, the microbiome shows lower levels of neuroprotective *Lactobacillus* [144]. Nonetheless, this decrease of the genus has not yet been directly associated with the development of ADHD. Much more important seems to be the combination of premature infants having underdeveloped brain structures and an immature immune system resulting in being more prone to neuronal cell death and infections that promote neuroinflammation, and finally influence the neurodevelopment. It is difficult to deduce the exact impact of microbial changes in preterms on the development of ADHD, as there are numerous confounders [153]. Thus, future studies should elucidate and concentrate on levels of pro-inflammatory cytokines in neonates and determine the extent to which underdeveloped brain structures influence the development of ADHD. Once these have been thoroughly understood, one can assess in what way the microbiome plays a role in the pathophysiology of prenates having a higher prevalence of ADHD. As the topic of this literature review is relatively new, only a limited number of studies examining the link between ADHD and the microbiota could be found. Hence, it was challenging to draw concrete conclusions from the scarce available data. A solid conclusion will require future investigations enrolling larger populations with defined pathologies to be able to analyze the study outcomes using robust statistical analysis. Finally, it is important that future trials use standardized methodologies for an unambiguous comparison of the outcomes and results. This literature review has made it clear that certain factors are associated with ADHD, while simultaneously changing the guts microbiome. Nevertheless, it remains yet to be determined to what extent the composition of the microbiome in the gut influences the development of ADHD.
