Clostridium bolteae

Clostridium histolyticum

C. histolyticum

C. perfringens

p

Candida albicans

Finegold et al. [ 12 ] in 2002 performed a series of microbiological studies of the intestinal content of autistic individuals and compared their results to those obtained in control children, with the hope of finding a unique and characteristic flora to the disorder. To the best of our knowledge, this report was the first demonstrating the existence of nine clostridial species in fecal samples from children with autism compared with only three found in control children. Non-clostridial anaerobes and microaerophilic bacteria were common in upper gastrointestinal specimens of autistic children but were not detected in healthy subjects. Perhaps, the main limitation of the study resides on its methodology of culture-based methods, which sometimes lead to significant underestimation of bacteria present in fecal samples. Since the authors were aware about the need to improve the sensibility of their approach, two years later, Song and collaborators (same research group) [ 13 ] studied the composition of intestinal flora based on the detection of rRNA genes (Real-Time PCR Quantitation, RT-PCR). Their analysis by RT-PCR showed that cell count differences between autistic children and healthy subjects forand the following Clostridium groups were statistically significant. Nevertheless, the authors indicated the need for further confirmations in large numbers of samples. Since numerical differences in the gut flora of ASD patients and healthy subjects needed further research, in 2005, Parracho et al. [ 14 ] performed an analysis of fecal samples from 58 children with autism by fluorescent in situ hybridization (FISH) using group-specific oligonucleotide probes and compare it with two healthy control groups (a non-autistic sibling group and an unrelated healthy group. Their data reported a higher incidence of the toxin producergroup (Clostridium clusters I and II) when compared to healthy children. However, these results differed for the second control group (non-autistic sibling) where intermediate level of thegroup was observed with no significant differences from any other analyzed groups. These results not only support the hypothesis of a link between clostridia and the development of certain autistic features but also suggest a putative therapeutic approach of autistic symptoms based on the modulation of gut microflora by reducing the numbers of Clostridium bacteria in ASD patients, while stimulating more beneficial gut bacteria (e.g., Lactobacillus or Bifidubacterium). Nonspecific symptoms are often related with intestinal inflammation and elevated level of fecal lactoferrin (FLA) resistant to proteolysis and bacteria degradation [ 15 ]. Martirosian et al. successfully found higher levels of FLA in stools from autistic children when matched with healthy controls [ 16 ] and even though toxins were not detected,were isolated significantly often from the autistic samples. In particular, intermediate sensitive strains to penicillin 19%, to clindamycin 11.3%, and to metronidazole 7.5%. Unfortunately, the studied groups were not big enough and additional confirmation of these data with larger groups was recommendable and the elucidation potential mechanism contributing to the negative consequences associated with ASD by studying the role of the gut microbiome and metabolome, considering that metabolome refers to the complete set of small-molecule chemical found within a biological sample if diverse origins. De Angelis et al. (2013) followed this approach [ 17 ] and compared total and active fecal microbiota from autism, PDD-NOS patients, and healthy controls through a culture-dependent and -independent methods as well as metabolomic analyses. Caloramator, Sarcina and Clostridium genera were the highest in autistic children. Compared to healthy individuals, the composition of Lachnospiraceae family was also especially different in the autistic group and PDD-NOS. In addition, phenol compounds such as phenol, 4-(1,1-dimethylethyl)-phenol, p-cresol) and free amino acids (Asp, Ser, Glu, Gly, Ala, Val, Ile, Phe, His, Tpr, Lys and Pro) were elevated in the feces of PDD-NOS and even more in samples from autistic subjects. These results are therefore consistent with the previous reports [ 12 16 ], suggesting that certain microbial and metabolic profiling could represent signatures for ASD, and invites for further investigation regarding additional markers and the pathophysiological roles of intestinal dysbiosis in these children. Xiong et al. [ 18 ] proposed a gas chromatography/mass spectrometry-based urinalysis to identify the metabolomic profile of these patients and demonstrated higher (< 0.001) concentrations 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA), 3-hydroxyphenylacetic acid (3HPA), and 3-hydroxyhippuric acid (3HHA) are present in these subjects when compared to healthy controls. Such values significantly decreased once these patients were submitted to vancomycin treatment indicating their Clostridium species-derived origin and suggesting these markers as putative predictors of ASD. In general, intestinal dysbiosis could be related to (i) yeast infection or (ii) leaky gut. One year later, Iovene and collaborators [ 19 ] searched for evidence demonstrating the presence of gastrointestinalfinding higher counts in autistic samples by using a diagnostic cultural approach. Nevertheless, it is unclear whether pharmacological-based therapies to eliminate yeasts in this patient could be useful to improve the behavioral hallmarks of the disorder.