Incidence of Clostridium perfringens and its toxin genes in the gut of children with autism spectrum disorder

doi:10.1016/j.anaerobe.2019.102114

Anaerobe

Volume 61, February 2020, 102114

https://doi.org/10.1016/j.anaerobe.2019.102114 Get rights and content

Highlights

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ASD group had a significantly higher incidence of C. perfringens than control group.
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The highest incidence of C. perfringens was found in the ASD group with GI symptoms.
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Beta-2 toxin gene (Cpb2) was present at significantly lower levels (25%) in the ASD group without GI symptoms.
•
High incidence of C. perfringens and its toxin gene (Cpb2) are associated with the GI complications in ASD which may affect the severity of the disease.
•
C. perfringens isolated from the ASD group were significantly more resistant to clindamycin.

Abstract

This study was designed to determine the incidence of Clostridium perfringens and their toxin genes in children with autism spectrum disorder (ASD), and determine the antimicrobial susceptibility of C. perfringens isolates. A hundred and fourteen fecal samples were obtained from children aged 3–12 years old (57 samples from ASD children and 57 from healthy controls). Children were divided into four groups based on their gastrointestinal (GI) symptoms as follows: ASD group with and without GI symptoms, and control group with and without GI symptoms. Selective anaerobic media and VITEK®2 ANC ID card were used for isolation and identification of C. perfringens from fecal samples. Antimicrobial susceptibility of C. perfringnes isolates were performed using (E-Test) strips against clindamycin, penicillin and metronidazole antibiotics. Conventional PCR was used to detect the alpha toxin gene (Cpa) and the beta-2 toxin gene (Cpb2). Genetic Analyzer 3130Xi was used to confirm the sequencing of Cpb2 gene. Our findings indicated that 38.6% of ASD group samples had a significantly (p = 0.003) higher incidence of C. perfringens than that of the control group (14%). The highest incidence of C. perfringens was found in the ASD group with GI symptoms (53.8%; p = 0.001). C. perfringens isolated from the ASD group (54.5%) were significantly (p = 0.047) more resistant to clindamycin than those isolated from the control group (12.5%). The C. perfringens isolates from the ASD and the control group showed 95.5% and 100% susceptibility to penicillin, respectively. All C. perfringens isolates of ASD and control group were susceptible to metronidazole. The Cpa toxin gene was also detected in all the C. perfringens isolates of ASD and control group, both with and without GI symptoms. Cpb2 toxin gene showed 100% incidence in ASD samples with GI symptoms and in the control groups both with or without GI symptoms, while it was present at significantly lower levels (25%) in the ASD samples without GI symptoms (p = 0.001). Our findings suggests that a high incidence of C. perfringens and its toxin gene (Cpb2) are associated with the GI complications in ASD which may affect the severity of the disease.

Introduction

Infectious agents have been recently taken into consideration with respect to the occurrence of autism spectrum disorder (ASD) [1]. Studies have shown that higher incidences of Clostridium sp. have been detected in the fecal matter of children with ASD than in healthy children [[2], [3], [4]]. Severe gastrointestinal (GI) symptoms in ASD children are probably due to the disruption in the gut flora, which leads to an overgrowth of pathogenic bacteria, and the production of harmful metabolic substances that may have an effect on the mitochondria [4,5].

Some strategies have been suggested to reduce the GI symptoms in ASD children, such as improving their gut microbiota profile through modulation of diet or decreasing the clostridial population levels [4]. A previous study has reported the regression of autism symptoms after using oral vancomycin, hypothesizing that decreasing the levels of Clostridium sp. may produce noteworthy improvements in the symptomatic treatment of ASD children [6]. Significantly higher level of C. perfringens were detected in the fecal matter of ASD children compared to healthy subjects [7].

C. perfringens has been associated with various serious diseases in both humans and animals [8,9]. C. perfringens strains secrete more than 20 extracellular toxins or hydrolytic enzymes, which may be the major virulence factors implicated in the pathogenicity of C. perfringens [10,11]. Conventional PCR results showed that the beta-2 toxin gene of C. perfringens (Cpb2) is significantly higher levels in ASD subjects than in healthy subjects [1,12,13]. The main aim of this study was the detection of the levels of C. perfringens and its toxin genes in the gut microbiota of children with ASD and comparison with those in children without ASD.

Section snippets

Subjects characteristics

This study was approved by the Scientific Research Ethics Committee of King Saud University, Riyadh, Saudi Arabia (Ref. No: KSU-SE-18-04) and parental consents were taken prior to the conduct of the study. Children aged 3–12 years old were recruited to participate in this study (57 children with diagnosed ASD and 57 healthy children as controls). The children were divided into four groups based on their GI symptoms as follows: Group I: ASD children with GI symptoms, Group II: ASD children

Results

The details regarding gender, age, and GI symptoms are summarized in Table 2. Our results indicated that 38.6% of the ASD samples (22/57) had significantly higher incidence of C. perfringens compared to the control group (14%; 8/57) (p =  0.003). Within the subgroups, the highest incidence of C. perfringens was found among the ASD group with GI symptoms (53.8%, 14/26; p = 0.001) (Fig. 1).

The gene encoding the alpha toxin, Cpa, was detected in all of the C. perfringens isolates from the ASD and

Discussion

A recent study reported that ASD children are four times likely to get GI complications compared to healthy children [18]. Investigations of gut microbiota as a contributing factor to GI complications and severity of symptoms in children with ASD have increased in recent times [18].

In the current study, we found that the GI symptoms occur in 45.6% of the ASD cases, compatible with previously reported GI symptoms prevalence in ASD children of 9%–90% [4,19,20]. Rigid-compulsive behavior,

Conclusion

In conclusion, high incidence of C. perfringens and its toxin gene, Cpb2, are associated with the GI complications in ASD, which may affect the severity of the ASD. However the role of C. perfringens and their toxins in ASD remain unclear. Therefore, further studies are required to elucidate the effects of C. perfringens and their toxins on ASD subject.

Declaration of competing interest

None of the authors have any conflict of interest to declare.

Acknowledgement

The authors would like to thank Deanship of scientific research for funding and supporting this research through the initiative of DSR Graduate Students Research Support (GSR).

References (34)

B. Góra et al.
Toxin profile of fecal Clostridium perfringens strains isolated from children with autism spectrum disorders
Anaerobe
(2018 Jun 1)
G. Martirosian et al.
Fecal lactoferrin and Clostridium spp. in stools of autistic children
Anaerobe
(2011 Feb 1)
S.M. Finegold et al.
Detection of Clostridium perfringens toxin genes in the gut microbiota of autistic children
Anaerobe
(2017 Jun 1)
S. Jeverica et al.
Evaluation of the routine antimicrobial susceptibility testing results of clinically significant anaerobic bacteria in a Slovenian tertiary-care hospital in 2015
Anaerobe
(2017 Oct 1)
R. Patusco et al.
Role of probiotics in managing gastrointestinal dysfunction in children with autism spectrum disorder: an update for practitioners
Adv. Nutr.
(2018 Sep 1)
R.G. Labbé et al.
Clostridium perfringens gastroenteritis
M. Gibert et al.
Beta 2 toxin, a novel toxin produced by Clostridium perfringens
Gene
(1997 Dec 5)
S.M. Finegold et al.
Gastrointestinal microflora studies in late-onset autism
Clin. Infect. Dis.
(2002 Sep 1)
Y. Song et al.
Real-time PCR quantitation of clostridia in feces of autistic children
Appl. Environ. Microbiol.
(2004 Nov 1)
H.M. Parracho et al.
Differences between the gut microflora of children with autistic spectrum disorders and that of healthy children
J. Med. Microbiol.
(2005 Oct 1)

R.E. Frye et al.

Gastrointestinal dysfunction in autism spectrum disorder: the role of the mitochondria and the enteric microbiome

Microb. Ecol. Health Dis.

(2015 Dec 1)

R.H. Sandler et al.

Short-term benefit from oral vancomycin treatment of regressive-onset autism

J. Child Neurol.

(2000 Jul)

K. Sim et al.

Dysbiosis anticipating necrotizing enterocolitis in very premature infants

Clin. Infect. Dis.

(2014 Oct 23)

F.H. Heida et al.

A necrotizing enterocolitis-associated gut microbiota is present in the meconium: results of a prospective study

Clin. Infect. Dis.

(2016 Jan 19)

S.A. Revitt-Mills et al.

Clostridium perfringens extracellular toxins and enzymes: 20 and counting

Microbiology (Australia)

(2015 Sep 17)

R. Kiu et al.

An update on the human and animal enteric pathogen Clostridium perfringens

Emerg. Microb. Infect.

(2018 Dec 1)

H.T. Ding et al.

Gut microbiota and autism: key concepts and findings

J. Autism Dev. Disord.

(2017 Feb 1)

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In previous studies, in Taiwan, Jordan, Egypt, Sweden, and Canada, the positive rate of cpe in isolates from chicken farms was 0% [5,20,23]. Beta-2 toxin encoded by the cpb2 gene, which can be produced by all types of C. perfringens, is associated with gastrointestinal disease in humans and animals [26]. In this study, the cpb2 positive rate of isolates from five farms was different, among which the highest positive rate was found in Farm 4 (64.46%, 78/121), and the lowest was found in Farm 5.
Clostridium perfringens (C. perfringens) is a common pathogenic microorganism present in nature, which can cause animal and human diseases, such as necrotizing enteritis (NE) in poultry. Little is known about the current prevalence status of C. perfringens from poultry farms of different types and regions in China. From December 2018 to August 2019, we investigated the prevalence, genotype distribution and drug resistance of C. perfringens from Guangdong, Pingyin, Tai'an and Weifang. A total of 622 samples were collected and processed for C. perfringens isolation, among which 239 (38.42%) samples were determined to be positive for C. perfringens. A total of 312 isolates of C. perfringens were recovered (1–5 strains were isolated for each positive sample), and 98.72% of the isolates were identified as type A, while the others were type F. Antimicrobial susceptibility testing revealed that 47.71% of the isolates were resistant to at least five classes of commonly used antibiotics. Multilocus sequence typing (MLST) showed that 74 representative isolates were divided into 63 sequence types (STs), and the Simpson's diversity index (Ds) of the STs for the five farms was 0.9799. 37.84% of the isolates were classified into seven clonal complexes (CC1–CC7), and the isolates from the same farm were more concentrated in the minimum spanning tree. In addition, some cloaca isolates and feed isolates were distributed in the same ST or CC; this result indicates that the C. perfringens in chicken can come from the environment (feed etc.).
Fermented Maillard reaction product alleviates injurious effects in colon caused by Clostridium perfringens
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C. perfringens causes not only foodborne illness, but bacteria-mediated infection, diarrhea, and necrotizing enterocolitis (Kiu & Hall, 2018; Lee & Yoon, 2021). It was discovered that the overabundance of C. perfringens in gut microbiota might cause neuromyelitis optica and autism (Cree et al., 2016; Alshammari et al., 2020). On the other hand, C. perfringens can inhabit the gastrointestinal tract asymptomatically.
Clostridium perfringens is an important bacterium among commensal bacteria in the gastrointestinal tract, which is known to possess potential pathogenicity during its inhabitation. In concordance with the concerns raised by its pathogenicity, this study aimed to elucidate if FMRP (fermented Maillard reaction products), the novel milk-derived material, can reduce the effect caused by the overabundance of C. perfringens in the gut. As a result, FMRP significantly reduced mRNA expression levels in the inflammatory cytokines (interleukin-10, interleukin-11, and interferon-γ) (p<0.05), and tumorigenic potential by activating the cell proliferation-suppressing factors (β-catenin, adenomatous polyposis coli, and E-cadherin) (p<0.05) and deactivation of the cell proliferation-inducing factor (cyclooxygenase-2) (p<0.05) in the colon. Moreover, the tight junction proteins (zonula occludens-1 and claudin-3) were recovered (p<0.05), and the antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) were decreased by FMRP (p<0.05). However, biochemical analyses showed no significant difference. These results indicate that C. perfringens might cause inflammation, oxidative stress, disruption of tight junctions and abnormal cell proliferation to the colon, but FMRP may reduce inflammation and generation of reactive oxygen, and recover tight junction in the colon.
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Furthermore, the relationship between Clostridium tetani sub-acute infection and ASD was hypothesized [242]. Similarly, association between the presence of beta2-toxin gene (produced by Clostridium perfringens) in the gut microbiome and development of ASD was reported [243]. Excessive use of antibiotics may result in the overgrowth of Desulfovibrio, i.e., bacteria resistant to common therapy, which release LPS [244].
Emerging evidence indicates that the gut microbiota play a crucial role in the bidirectional communication between the gut and the brain suggesting that the gut microbes may shape neural development, modulate neurotransmission and affect behavior, and thereby contribute to the pathogenesis and/or progression of many neurodevelopmental, neuropsychiatric, and neurological conditions. This review summarizes recent data on the role of microbiota-gut-brain axis in the pathophysiology of neuropsychiatric and neurological disorders including depression, anxiety, schizophrenia, autism spectrum disorders, Parkinson’s disease, migraine, and epilepsy. Also, the involvement of microbiota in gut disorders co-existing with neuropsychiatric conditions is highlighted. We discuss data from both in vivo preclinical experiments and clinical reports including: (1) studies in germ-free animals, (2) studies exploring the gut microbiota composition in animal models of diseases or in humans, (3) studies evaluating the effects of probiotic, prebiotic or antibiotic treatment as well as (4) the effects of fecal microbiota transplantation.
Epigenetic upregulation of ssc-miR-124a following treatment with Clostridium perfringens beta2-toxin attenuates both apoptosis and inflammation in intestinal porcine epithelial cells
2021, Archives of Biochemistry and Biophysics
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Previous studies have revealed that over 97% of C. perfringens isolates from pig diarrhea could produce beta2-toxin [7], in addition to C. perfringens strains producing alpha toxin. Moreover, a high presence rate of beta2-toxin gene has been associated with gastrointestinal diseases in human [27], chicken [28], various ruminants [29,30] and horses [31]. Manteca et al. [32] reported that in vivo synergistic role of the beta2 and alpha toxins of C. perfringens in the production of necrotic and haemorrhagic lesions of the small intestine in the bovine enterotoxemia syndrome.
Clostridium perfringens (C. perfringens) is a globally recognized zoonotic pathogen. It has been reported that the beta2-toxin produced by C. perfringens can cause a variety of gastrointestinal diseases and even systemic inflammation. MicroRNA-124a (miR-124a) has been reported to play important roles in the host response to pathogenic infection. Although C. perfringens beta2-toxin induced injury in intestinal porcine epithelial (IPEC-J2) cells has been established, the underlying molecular mechanism is not completely unraveled. Here we show that a significant upregulation of ssc-miR-124a in IPEC-J2 cells after beta2-toxin stimulation was associated with the MiR-124A-1 and MiR-124A-2 gene promoter demethylation status. Importantly, overexpression of ssc-miR-124a significantly increased cell proliferation and decreased apoptosis and cytotoxicity in beta2-toxin treated IPEC-J2 cells. Transfection of IPEC-J2 cells with ssc-miR-124a mimic suppressed beta2-toxin induced inflammation. On the contrary, ssc-miR-124a inhibitor promoted aggravation of cell apoptosis and excessive damage. Furthermore, rho-associated coiled-coil-containing protein kinase 1 (ROCK1) was identified as the direct target gene of ssc-miR-124a in IPEC-J2 cells and its siRNA transfection reversed the promotion of apoptosis and aggravation of cellular damage induced by ssc-miR-124a inhibitor. Overall, we speculated that the miR-124A-1/2 gene was epigenetically regulated in IPEC-J2 cells after beta2-toxin treatment. Upregulation of ssc-miR-124a may restrain ROCK1, and attenuate apoptosis and inflammation induced by beta2-toxin that prevent IPEC-J2 cells from severe damages. We discover a new molecular mechanism by which IPEC-J2 cells counteract beta2-toxin‐induced damage through the ssc-miR-124a/ROCK1 axis partially.

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Clostridioides (Clostridium) difficile (including epidemiology)Incidence of Clostridium perfringens and its toxin genes in the gut of children with autism spectrum disorder

Highlights

Abstract

Introduction

Section snippets

Subjects characteristics

Results

Discussion

Conclusion

Declaration of competing interest

Acknowledgement

Anaerobe

Anaerobe

Anaerobe

Anaerobe

Adv. Nutr.

Gene

Gastrointestinal microflora studies in late-onset autism

Clin. Infect. Dis.

Real-time PCR quantitation of clostridia in feces of autistic children

Appl. Environ. Microbiol.

Differences between the gut microflora of children with autistic spectrum disorders and that of healthy children

J. Med. Microbiol.

Gastrointestinal dysfunction in autism spectrum disorder: the role of the mitochondria and the enteric microbiome

Microb. Ecol. Health Dis.

Short-term benefit from oral vancomycin treatment of regressive-onset autism

J. Child Neurol.

Dysbiosis anticipating necrotizing enterocolitis in very premature infants

Clin. Infect. Dis.

A necrotizing enterocolitis-associated gut microbiota is present in the meconium: results of a prospective study

Clin. Infect. Dis.

Clostridium perfringens extracellular toxins and enzymes: 20 and counting

Microbiology (Australia)

An update on the human and animal enteric pathogen Clostridium perfringens

Emerg. Microb. Infect.

Gut microbiota and autism: key concepts and findings

J. Autism Dev. Disord.

Clostridioides (Clostridium) difficile (including epidemiology)
Incidence of Clostridium perfringens and its toxin genes in the gut of children with autism spectrum disorder