The Maternal/Fetal, Newborn Exposure to Certain Antibiotics, Might Promote ASD-Related Dysbiosis in European Countries; Supporting Experimental Observations

1. Introduction

Autism spectrum disorder (ASD), which was first described by Karner (1942) is a neurodevelopmental disease, which is characterized by deficits in social communication and the presence of restricted interests and repetitive behaviors [1]. Its recent, pandemic-like spread is alarming and it became a public health issue. According to the Centers for Disease Control and Prevention (CDC) report in 2018, the ASD prevalence among children by 8 years has been estimated as 16.8/ 1000 (1:56) with more boys (26.6/ 1000 boys) than girls (6.6/ 1000 girls), which means a 150% increase from 2000 to 2014 [2]. The World’s prevalence of ASD has increased several folds since the first epidemiological observations in the past ~50 years and has reached the level of 1-2% of all children by 2000. The reasons for the increase could not be clearly stated, but researchers agree that better diagnostic criteria and some external factors might play a role [2].

People did not know much about autism before the excellent film of Rain man, but the pandemic of autism is rapidly spreading and by now, 1 in 56 children have a chance to develop autism and we are still not capable to show up any appropriate reason for this ailment, despite the fact, that several associations of different conditions were suspected as a causative agent [3,4]. Genetic mechanisms account for approximately 10–20% of ASD cases [5,6]. It was observed, that vancomycin treatment ameliorated the symptoms of ASD, suspecting the role of some clostridial bacteria [7,8,9]. The abundance of Clostridium bolte in autism prompted speculation about the use of developing a vaccine against this pathogen [10]. Desulfivibrio was suspected as an etiological agent in autism also [11]. Several publications support the role of intestinal bacteria [12,13]. The role of transvaginal ultrasonography [14], and conjugate vaccines [15] accumulation of insulin-like growth factors [16]. Other reasons leading to autism were also discussed in the literature [17]. A few of the latest proposal includes the role of electromagnetic frequency and radiofrequency radiation exposures (EMF/RFR) [18]. Tylenol/Augmentin and pesticide combined are suspected also [19]. Moreover, new evidence suggests that the peripheral immune system plays an important role in normal neuronal function [20,21,22].

ASD comprises a group of developmental disorders, starting in early childhood and characterized by pervasive behavioral deficits and social interaction [23]. The International Classification of Diseases and the Diagnostic Statistical Manual that the ASD group includes childhood autism, atypical autism, Asperger’s syndrome, disintegrative disorders, and Rett syndrome. ASD is considered genetically and phenotypically as a heterogenous group of symptoms with various severity, symptomatology, and outcome [24]. All manifestations are considered serious, devastating manifestations regarding the outcome, and co-morbidity, which bears down the affected families, society, and the health care systems alike. The overlap between autism and childhood obesity indicates that autistic patients are obese as well. Recent publications in increasing numbers indicate the role of microbiome alteration (dysbiosis) in autism. Experiments proved that fecal samples from autistic children when injected into germ-free mice, produce similar symptoms [25,26].

In ASD cases the balance between the pro-inflammatory bacteria (clostridia and desulfivibrio) and the anti-inflammatory bacteria (bifidobacteria) destabilized even before the symptoms of ASD occurred. This imbalance results in the so-called “leaky gut” syndrome and through the more porous epithelial membrane, toxins, produced by the microbes, are easily can enter the circulation, which may affect the brain and the development of ASD [27,28]. The alteration of the intestinal microbial taxa in ASD cases is repeatedly reported as the reduction of Firmicutes and an increase in the number of Bacteroidetes [29].

Other investigators observed the relative abundance of other bacteria, like Clostridia, Caloramator, Alistipes, Sarcina, Akkermansia, Lactobacillus, Enterobacteriaceae, and Sutterellaceae [30,31,32,33,34], and the reduction in the abundance of Bifidobacteria, Desulfovibrio, Coprococcus, Veillonellaceae and Prevotellaare species [8]. The pathogenetic process of ASD might be associated with the overgrowth or the reduction of different species, like Clostridium, which develops particularly with the use of antibiotics [11,35,36,37,38,39].

The presence of an altered microbiome (dysbiosis) in ASD is well documented and its possible role in the development of ASD has been reported also (see above references). It was hypothesized that antibiotic exposure to the microbiome might produce “ASD-promoting” dysbiosis in the mother, or the early modification of the gut microbiome in infancy might facilitate the development of ASD through the action of different mediator molecules involving the gut-brain axis (GBA) [40].

In mice, in utero exposure to the anticonvulsant valproic acid (VPA), leads to developmental and behavioral deficits in offspring that are similar to ASD [41].

Recent studies indicated two specific microbiota-derived host metabolites, p-cresol sulfate, and 4-ethyl phenyl sulfate, have been associated with ASD in both patients and animal models. These metabolites originate from bacterially produced p-cresol and 4-ethylphenol, respectively, which are produced through aromatic amino acid fermentation by a range of commensal bacteria, most notable bacteria from the Clostridioides genus, which are among the dysregulated bacteria frequently detected in ASD patients [42].

Epidemiological studies have revealed that early-life antibiotic exposure can increase the risk of neurodevelopmental disorders later in life [43,44].

Hypothesis

It is of consideration that different classes of antibiotics might induce different dysbiosis; hence, different molecular products of the altered microbiome leaking in the circulation might augment or inhibit the development of ASD. We have aimed to identify antibiotic classes, which might promote or inhibit the development of ASD-related dysbiosis and hence the prevalence of ASD.

2. Materials and methods

To evaluate the above hypothesis, antibiotic consumption databases were compared to the autism (ASD) prevalence in countries of people living with autism/ 100000 population in 30 European countries estimated for 2023 (https://wisevoter.com/country-rankings/autism-rates-by-country/).

Average, yearly antibiotic consumption had been calculated from the publicly available antibiotic databases of 30 European countries (ECDC yearly reports) for 1997-2020 https://www.ecdc.europa.eu/en/antimicrobial-consumption/database/quality-indicators. Based on the Anatomical Therapeutic Chemical classification system (ATC), the average yearly consumption of the total systemic antibiotics (J01) has been calculated and expressed in Defined Daily Dose/1000 Inhabitants/ Day (DID). The consumption of major antibiotic classes covering the 94% antibiotic use in the community at ATC levels two and three were calculated as a relative share of the total amount of systemic antibiotics (J01) and expressed in percentage (%). Antibiotic classes included: tetracycline (J01A), penicillin (J01C), broad-spectrum, beta-lactamase sensitive penicillin (J01CA), narrow spectrum, beta-lactamase sensitive penicillin (J01CE), narrow spectrum, beta-lactamase resistant penicillin (J01CF), broad-spectrum, beta-lactamase resistant combination penicillin (J01CR), cephalosporin (J01D), macrolide and lincosamides, streptogramins (J01F), and quinolone (J01M) expressed in percentage of the total amount.

Statistics

Pearson calculation was applied to estimate the correlation between antibiotic consumption and the prevalence of ASD. A significant correlation (positive/negative) was considered when p values were ≤ 0.05. A non-significant correlation was estimated when the p values fall between 0.051-0.09. Positive (supportive) and negative (inhibitor) significant correlations were considered and evaluated. Logistic regression analysis was performed to determine the odds ratio (OR, CI 95%) for each antibiotic class. A significant result was estimated when p values were equal to or less than 0.05 (≤ 0.05). A non-significant positive/negative) correlation has been considered when the p-value falls between 0.05 and 0.09 (p: 0.05 ≤ 0.09). The Kruskal–Wallis test was performed to determine statistically significant differences among the groups formed by country ranking. A significant difference was considered when p values were equal to or less than 0.05 (≤ 0.05), and a nonsignificant difference was considered when p values were between 0.051–0.09. Statistical results were recorded and featured in Table 1. Scatter diagrams were plotted to demonstrate the association (positive/negative) between the prevalence of autism and the average consumption of narrow spectrum, beta-lactamase resistant penicillin (J01CF) and tetracycline (J01A), (Figure 1 and Figure 2).

3. Results

Tetracycline (J01A) consumption showed a significant positive (promoting) association with the prevalence of ASD (Pearson r: 0.373, p: 0.043. OR: 1.312, CI95%: 0.995-1.791, p: 0.065) and narrow-spectrum, beta-lactamase resistant penicillin (J01CF) (Pearson r: 0.524, p: 0.003, OR: 3.240, CI95%: 1.710-8.853, p: 0.004, Kruskal-Wallis p: 0.032). Mild, negative (inhibitory) association was observed with broad-spectrum, beta-lactamase sensitive penicillin (J01CA) (Pearson r: -0.278, p: 0.157, OR: 0.808, CI95%: 0649-0957, p: 0.028) and narrow-spectrum, beta-lactamase-sensitive penicillin (J01CE) (Pearson p: -0.032, r: 0.865, OR: 0.725, CI95%: 0.543-0.885, p: 0,009).

4. Discussion

Early life is a critical period for neurodevelopment. In recent decades, there has been a rise in the incidence of childhood neurodevelopmental disorders including ASD, attention-deficit/hyperactivity disorder (ADHD), and learning disabilities worldwide. It has been observed that the incidence of ASD increased significantly over time, especially among toddlers and preschool children but also in older age groups [45]. Recent publications (cited above) agree on the principal role of certain gut bacteria in the development of autism, particularly of clostridia species. This theory was supported by the fact that vancomycin ameliorated the symptoms of autism, but it returned after the cessation of antibiotic treatment, probably indicating that after temporally suppressing the clostridial species, the appropriate balance of the gut flora was not restored [46]. Similarly, the fecal microbiome transfer has greatly ameliorated the symptoms of ASD [47,48,49]. The initial development and maturation of the neonatal microbiome are largely determined by maternal–offspring exchanges of microbiota, which indicates that dysbiotic maternal microbiome might populate the newborn intestine also [50].

The importance of biochemical changes observed early in the newborn, such as the depressed level of insulin-like growth factor-1 (IGF-1) in the process of the neurodevelopmental period, are considered important by several researchers in the development of ASD. This observation leads to early diagnosis and probable intervention in the prevention of developing dysconnectivity. According to Steinmann [51], the process of developing ASD could be preventable even before irreversible psychosocial changes develop.

It might be concluded that biochemical changes observed in ASD, are probably triggered by the altered microbiome induced by antibiotics. Reports on the association between autism and antibiotics are controversial. Some observations indicate a certain association between prenatal exposure to antibiotics and autism [52], while others found some beneficial effects of perinatal antibiotic use [53]. Controversy might arise from the fact that different antibiotic classes induce different dysbiosis, which either promotes or inhibits the development of ASD-related dysbiosis.

In animal experiments, when newborn mice were exposed to low-dose penicillin, profound changes in the intestinal microbiota were observed, which might be implicated in the perturbance of neurodevelopmental and neuropsychiatric pathways. Significant effects were observed on different areas of the brain (frontal cortex, amygdala gene, etc.). Linkage was observed between the specific microbial taxa and the early-life expression of particularly affected genes [54].

The results derived from animal models [55] strongly support our observations as we have detected the possible association between the consumption of the narrow-spectrum, beta-lactamase-resistant penicillin (J01CF) and the prevalence of autism, which might induce developmental difficulties in the brain, resulting in ASD. Group B Streptococcus (GBS) remains the most common cause of neonatal early-onset sepsis among term infants and a major cause of late-onset sepsis among both term and preterm infants and penicillin is frequently used for prophylaxis [56].

Our comparative analyses indicated a significant, positive correlation between the consumption of tetracycline/penicillin and the prevalence of ASD. It was observed that the highest rate of ASD and the highest tetracycline (J01A) consumption together with the second highest consumption of narrow-spectrum, beta-lactamase resistant penicillin (J01CF) is reported from the UK. Similarly, Sweden records the highest consumption of narrow-spectrum, beta-lactamase-resistant penicillin (J01CF) and fifth place on the tetracycline consumption rank order and second place on the ASD prevalence list. (Table 1). As far as pregnant women, newborn babies, and infants are not taking any tetracycline compound because it is contraindicated, it could be suspected, that tetracycline might arrive from environmental antibiotic pollution, or the women were exposed to tetracycline before being pregnant, which triggered the ASD-related dysbiosis. As a medication, more than 70 % of tetracycline antibiotics are excreted and released in active form into the environment via urine and feces from humans and animals. Tetracycline has become a serious threat to the environment because of its overuse by humans and veterinarians and its weak ability to degrade. Tetracycline is capable of accumulating along the food chain and influencing microbial communities [57]. It might be concluded that higher consumption could result in higher environmental pollution also.

Clinical and experimental data indicate that the risk of developing ASD is associated with the presence of inflammatory changes during pregnancy and the modification of the microbiome that defined gut commensal bacteria with a propensity to induce Th17 cells may increase the risk for neurodevelopmental disorders in the offspring of pregnant mothers undergoing immune system activation due to infections or auto-inflammatory syndromes [58,59,60,61,62].

Animal models (mice) indicated that applying a low dose of Penicillin V induced a more robust behavioral abnormalities response than the cocktail of broad-spectrum antibiotics [63].

The above findings strongly support our observations regarding the possible role of narrow-spectrum penicillin (J01CF) as a possible putative factor in the development of ASD.

4.1. Limitations of our study

Our results could not be interpreted at the individual level, only statistical correlations and concordance could be established, but a firm, statistical correlation between tetracycline and penicillin consumption and ASD was estimated. This might indicate a possible association between maternal consumption of tetracycline/penicillin (dysbiosis) and autism, shown in the scatter diagrams also.

4.2. Strength of our study

The results of our comparative analysis is in full accord with previous animal experiments indicating the role of even low-dose penicillin applied intrauterine, or newborn mice, in the development of autism-like behavior.