Genetic variation among Clostridium perfringens isolated from food and faecal specimens in Lagos.

BACKGROUND: Clostridium perfringens is an anaerobic Gram-positive bacterium which is commonly present in the gastrointestinal tract of man and animals and causes enteritic diseases in animals and food poisoning in humans. Previous studies have looked at the epidemiological relationship between C. perfringens isolates from outbreak source. In this study, the genetic diversity of C. perfringens strains from non-outbreak food and faecal specimens was investigated for epidemiological purposes. METHODS: We analyzed thirty-eight (38) Clostridium perfringens strains isolated from food and faecal specimens in Lagos State. Bacterial identification was done using colonial morphology, Gram stain reaction, conventional biochemical tests and PCR. Genetic analysis was performed using arbitrary primed polymerase chain reaction (AP-PCR) technique with oligonucleotide primer of random sequences (OPA-3) to determine the genetic diversity of C. perfringens. The distance between the different bands produced were analyzed using numerical taxonomy and multivariate system software (NTSYS). RESULTS: Seventeen (44.7%) C. perfringens strains showed at least one polymorphic DNA patterns when genotyped. However, this method identified polymorphisms among the C. perfringens species from which four genetic groups (1, 2, 3 and 4) were established. CONCLUSIONS: Our findings suggest that there may be faecal contamination of food products and similar clones of Clostridium perfringens may be incriminated.

The aggravation of arthritis by periodontitis is dependent of IL-17 receptor A activation.

AIM: To evaluate whether Porphyromonas gingivalis-induced periodontitis aggravates the antigen-induced arthritis (AIA) model, and whether this effect is dependent on the Th17/IL-17 signalling pathway. MATERIALS AND METHODS: Antigen-induced arthritis was triggered by local injection of methylated bovine serum albumin into the knee joint of previously immunized C57BL/6 wild-type (WT) and IL-17 receptor A (IL-17RA)-knockout mice. Periodontal disease in naïve or arthritic mice was induced by oral infection with P. gingivalis. Animals were sacrificed 7, 15 and 30 days after infection. Alveolar bone loss, joint histopathology, articular hyperalgesia and joint cytokine production were assessed, in addition to the proportion of Th17 and Treg cells isolated from the inguinal lymph nodes. RESULTS: No influence of experimentally-induced arthritis was found on the alveolar bone resorption induced by P. gingivalis. However, mice with experimentally-induced arthritis that were exposed to P. gingivalis presented higher joint damage and Th17 frequencies when compared to non-infected mice. The aggravation of arthritis by periodontitis was accompanied by increased TNF and IL-17 production and articular neutrophil infiltration, whereas arthritis aggravation and changes in neutrophil infiltration were absent in IL-17RA-deficient mice. CONCLUSION: The effects of P. gingivalis-induced periodontitis on arthritis are dependent on Th17 expansion and IL-17RA signalling, which lead to increased neutrophil infiltration into the joints.

Periodontal pathogens directly promote autoimmune experimental arthritis by inducing a TLR2- and IL-1-driven Th17 response.

Increasing epidemiologic evidence supports a link between periodontitis and rheumatoid arthritis. The actual involvement of periodontitis in the pathogenesis of rheumatoid arthritis and the underlying mechanisms remain, however, poorly understood. We investigated the influence of concomitant periodontitis on clinical and histopathologic characteristics of T cell-mediated experimental arthritis and evaluated modulation of type II collagen (CII)-reactive Th cell phenotype as a potential mechanism. Repeated oral inoculations of periodontal pathogens Porphyromonas gingivalis and Prevotella nigrescens induced periodontitis in mice, as evidenced by alveolar bone resorption. Interestingly, concurrent periodontitis induced by both bacteria significantly aggravated the severity of collagen-induced arthritis. Exacerbation of arthritis was characterized by increased arthritic bone erosion, whereas cartilage damage remained unaffected. Both P. gingivalis and P. nigrescens skewed the CII-specific T cell response in lymph nodes draining arthritic joints toward the Th17 phenotype without affecting Th1. Importantly, the levels of IL-17 induced by periodontal pathogens in CII-specific T cells directly correlated with the intensity of arthritic bone erosion, suggesting relevance in pathology. Furthermore, IL-17 production was significantly correlated with periodontal disease-induced IL-6 in lymph node cell cultures. The effects of the two bacteria diverged in that P. nigrescens, in contrast to P. gingivalis, suppressed the joint-protective type 2 cytokines, including IL-4. Further in vitro studies showed that the Th17 induction strongly depended on TLR2 expression on APCs and was highly promoted by IL-1. Our data provide evidence of the involvement of periodontitis in the pathogenesis of T cell-driven arthritis through induction of Ag-specific Th17 response.

Detection of toxigenic Clostridium perfringens and Clostridium botulinum from food sold in Lagos, Nigeria.

Food-borne diseases contribute to the huge burden of sickness and death globally and in the last decade, have become more frequently reported in Africa. In line with this, food safety is becoming a significant and growing public health problem in Nigeria. Diarrhoea is a common problem in Nigeria and has been reported but there has been little data on the possibility of clostridia as aetiological agents. Clostridium species are ubiquitous in the environment and in the gastrointestinal tract of man and animals and can serve as a marker for faecal contamination. We set out to determine the potential of these foods to transmit Clostridium species. A total of 220 food commodities from six local governments in Lagos State were sampled. Isolates obtained were identified based on cultural, morphological and biochemical characteristics. Toxinotyping was done using multiplex-PCR with primers specific for alpha, beta, epsilon and iota-toxin genes, enterotoxigenic cpe gene and neurotoxigenic BoNt gene. Fifty (22.7%) clostridial species were isolated of which 29 (58%) were identified as C. perfringens. Toxinotyping of the 29 strains showed that 28 (96.6%) were toxin producing C. perfringens type A while one (3.4%) was C. perfringens type D. Two (4%) C. botulinum species were isolated and identified by 16S rRNA sequencing, both harbouring BoNt/A gene. The contamination rates of food with Clostridium species show that food hygiene is a problem and Clostridium species may be a source of food borne disease in Lagos State, Nigeria.

Sialidase production and genetic diversity in Clostridium perfringens type A isolated from chicken with necrotic enteritis in Brazil.

The sialidase activity and genetic diversity of 22 Clostridium perfringens strains isolated from chickens with necrotic enteritis were determined. Sialidase activity was detected in 86.4 % of the strains. All C. perfringens showed a high value of similarity (>96 %), and they were grouped into seven clusters clearly separated from the other reference bacterial strains. From these clusters four patterns were defined in accordance with their phenotypic (sialidase production and antibiotic resistance profile) and genotypic (presence of nanI and nanJ genes) characteristics. Our results showed heterogeneity among strains, but they were genotypically similar, and it is suggested further studies are needed to better understand the pathogenesis of necrotic enteritis.

Occurrence and antimicrobial susceptibility of Porphyromonas spp. and Fusobacterium spp. in dogs with and without periodontitis.

The occurrence of Porphyromonas gulae, Porphyromonas macacae, Fusobacterium nucleatum and Fusobacterium canifelinum in subgingival plaque from dogs with and without periodontitis as well as their antimicrobial susceptibility were evaluated. From 50 dogs with periodontitis were identified 38 P. gulae, 8 P. macacae, 26 F. nucleatum and 15 F. canifelinum, and from 50 dogs without periodontitis were identified 15 P. gulae, 12 F. nucleatum and 11 F. canifelinum. All strains were susceptible to most of the antibiotics tested, however, different resistance rates to clarithromycin, erythromycin and metronidazole among strains were observed. The role of P. gulae, P. macacae, F. nucleatum and F. canifelinum in periodontal disease of household pets needs to be defined to a better prevention and treatment of the canine periodontitis.

Detection of Porphyromonas gulae from subgingival biofilms of dogs with and without periodontitis.

A rapid PCR approach was developed to detect Porphyromonas gulae strains from subgingival samples of dogs with and with periodontitis. The presence of P. gulae was observed in 92% and 56%, respectively, in dogs with and without periodontitis. The new primer pair was specific to detect this microorganism, and this technique could be used to evaluate a correlation between periodontitis and P. gulae in companion animals.

Regulatory T cells attenuate experimental periodontitis progression in mice.

AIMS: The aim of this study was to identify the presence and characterize the function of regulatory T cells (Tregs) in experimental periodontitis in mice. MATERIAL AND METHODS: C57Bl/6 mice infected with Actinobacillus actinomycetemcomitans, treated or not with anti-glucocorticoid-inducible tumour necrosis factor receptor (anti-GITR) to inhibit Tregs function, were analysed regarding inflammatory cell and Tregs influx, alveolar bone loss and cytokine expression/production (analysed by real-time polymerase chain reaction and ELISA) throughout experimental periodontitis. RESULTS: A. actinomycetemcomitans inoculation in mice resulted in periodontal disease characterized by marked alveolar bone loss and an influx of inflammatory cells. Flow cytometry evaluation of inflammatory cells demonstrated an increased number of CD4(+)CD25(+) and CD4(+)FOXp3(+) cells, characterizing the presence of Tregs in the periodontal environment in a late stage after infection. Tregs-associated cytokines interleukin-10 (IL-10), cytotoxic T lymphocyte-associated molecule 4 (CTLA-4) and transforming growth factor-beta (TGF-beta) were found to be expressed/produced in a kinetics that resembles Tregs migration. Treatment with anti-GITR, which inhibits Tregs function, showed increased alveolar bone loss and inflammatory cell migration. A reduction in IL-10, CTLA-4 and TGF-beta levels was also observed, while interferon-gamma, tumour necrosis factor-alpha and receptor activator for nuclear factor kappaB ligand levels were increased. However, bacterial load and C-reactive protein serum did not show any differences. CONCLUSION: Taken together, our results showed that the presence of Treg cells attenuates the severity of experimental periodontitis without impairment in the control of infection.

Presence of periodontopathic bacteria in coronary arteries from patients with chronic periodontitis.

In this study the presence of periodontopathic pathogens in atheromatous plaques removed from coronary arteries of patients with chronic periodontitis and periodontally healthy subjects by PCR was detected. Our results indicate a significant association between the presence of Porphyromonas gingivalis and atheromas, and the periodontal bacteria in oral biofilm may find a way to reach arteries.

Prevalence of Enteropathogens and Virulence Traits in Brazilian Children With and Without Diarrhea.

The use of molecular diagnostics for pathogen detection in epidemiological studies have allowed us to get a wider view of the pathogens associated with diarrhea, but the presence of enteropathogens in asymptomatic individuals has raised several challenges in understanding the etiology of diarrhea, and the use of these platforms in clinical diagnosis as well. To characterize the presence of the most relevant bacterial enteropathogens in diarrheal episodes, we evaluated here the prevalence of diarrheagenic E. coli pathotypes, Salmonella spp., and Yersinia enterocolitica in stool samples of children with and without diarrhea using real-time quantitative PCR (qPCR). We found that the presence of genetic markers associated with bacterial pathogens was significantly higher in stool samples from the diarrhea group compared to the control (P < 0.001). Bacterial loads in samples positive for eae and aggR markers were also determined. Compared to samples from asymptomatic children, a significantly higher number of copies of the eae gene were found in diarrhea samples. Also, the presence of genetic markers associated with STEC strains with clinical significance was evaluated in eae-positive samples by high-throughput real-time PCR. The data presented herein demonstrated that asymptomatic children of an urban area in Brazil might be enteropathogen reservoirs, especially for STEC.

An interleukin-1beta (IL-1beta) single-nucleotide polymorphism at position 3954 and red complex periodontopathogens independently and additively modulate the levels of IL-1beta in diseased periodontal tissues.

Inflammatory cytokines such as interleukin-1beta (IL-1beta) are involved in the pathogenesis of periodontal diseases. A high individual variation in the levels of IL-1beta mRNA has been verified, which is possibly determined by genetic polymorphisms and/or by the presence of periodontopathogens such as Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacter actinomycetemcomitans. In this study, we investigated the role of an IL-1beta promoter single-nucleotide polymorphism at position 3954 [IL-1beta(3954) SNP] and the presence of the periodontopathogens in the determination of the IL-1beta levels in the periodontal tissues of nonsmoking chronic periodontitis (CP) patients (n = 117) and control (C) subjects (n = 175) and the possible correlations with the clinical parameters of the disease. IL-1beta(3954) SNP was investigated by restriction fragment length polymorphism, while the IL-1beta levels and the presence of the periodontopathogens were determined by real-time PCR. Similar frequencies of IL-1beta(3954) SNP were found in the C and CP groups, in spite of a trend toward a higher incidence of T alleles in the CP group. The IL-1beta(3954) SNP CT and TT genotypes, as well as P. gingivalis, T. forsythia, and T. denticola, were associated with higher IL-1beta levels and with higher values of the clinical parameters of disease severity. Concomitant analyses demonstrate that IL-1beta(3954) and the red complex periodontopathogens were found to independently and additively modulate the levels of IL-1beta in periodontal tissues. Similarly, the concurrent presence of both factors was associated with increased scores of disease severity. IL-1beta(3954) genotypes and red complex periodontopathogens, individually and additively, modulate the levels of IL-1beta in the diseased tissues of nonsmoking CP patients and, consequently, are potentially involved in the determination of the disease outcome.

The essential role of IFN-gamma in the control of lethal Aggregatibacter actinomycetemcomitans infection in mice.

Inflammatory immune reactions in response to periodontopathogens trigger periodontal destruction, but their role to protect the host against infection remains unknown. Thus, we examined the mechanisms by which IFN-gamma modulates the outcome of Aggregatibacter actinomycetemcomitans-induced periodontal disease in mice. Our results showed that IFN-gamma deficient mice developed less severe periodontitis in response to A. actinomycetemcomitans infection, characterized by significant lower alveolar bone loss and inflammatory reaction. However, the absence of IFN-gamma results in increased bacterial load in periodontal tissues and higher acute phase reaction, followed by a disseminated bacterial infection and mice death during the course of the disease. Such impaired host response was found to be associated with a reduction in the levels of inflammatory cytokines and chemokines and in the number of GR1+, F4/80+, CD4+ and CD8+ leukocytes in the diseased periodontium of IFN-gamma deficient mice. In addition, the levels of both antimicrobial mediators myeloperoxidase and inducible nitric oxide synthase were also found to be reduced in IFN-KO mice. Our results demonstrate for the first time that a periodontal infection may be lethal in an immunocompromised host. In addition, the mechanisms involved in IFN-gamma mediated cell migration to diseased periodontal tissues, and its essential role to control A. actinomycetemcomitans infection were clarified.

Occurrence of enterotoxigenic and nonenterotoxigenic Bacteroides fragilis in calves and evaluation of their antimicrobial susceptibility.

Bacteroides fragilis is considered an important clinical pathogen and the most common anaerobe isolated from human and animal clinical specimens; enterotoxigenic strains produce diarrhea. The presence of enterotoxigenic (ETBF) and nonenterotoxigenic B. fragilis in stool samples from calves with or without acute diarrhea and the antimicrobial susceptibility of the strains were evaluated. The stool samples were plated onto a selective B. fragilis-bile-esculin agar, and incubated anaerobically (10% CO(2)/90% N(2)), at 37 degrees C, for 72 h. Species of the B. fragilis group were identified by using the API 32-A kit. Enterotoxigenic strains were detected by PCR and the cytotoxic assay. From 54 diarrhea and 54 nondiarrhea stools, 124 and 92 members of the B. fragilis group, respectively, were recovered. Only two ETBF strains were isolated from two different diarrhea samples and the bft gene was detected in both. Moreover, the bft gene was detected in DNA from four different diarrheal stools samples but no ETBF strain was recovered. All the bacteria were susceptible to chloramphenicol, imipenem, moxifloxacin, piperacillin/tazobactam, metronidazole and tigecycline. Most of the isolates from both calves with and without diarrhea were resistant to all metals. Our results are of concern, and suggest the need to increase the surveillance of antibiotic and metal resistance of this microbial group isolated from animal production such as calves.

Adhesion and invasion of Clostridium perfringens type A into epithelial cells.

Clostridium perfringens is the causative agent for necrotic enteritis. It secretes the major virulence factors, and α- and NetB-toxins that are responsible for intestinal lesions. The TpeL toxin affects cell morphology by producing myonecrosis, but its role in the pathogenesis of necrotic enteritis is unclear. In this study, the presence of netB and tpeL genes in C. perfringens type A strains isolated from chickens with necrotic enteritis, their cytotoxic effects and role in adhesion and invasion of epithelial cells were evaluated. Six (27.3%) of the 22 C. perfringens type A strains were harboring the tpeL gene and produced morphological alterations in Vero cells after 6h of incubation. Strains tpeL (-) induced strong cell rounding after 6h of incubation and produced cell enlargement. None of the 22 strains harbored netB gene. All the six tpeL (+) gene strains were able to adhere to HEp-2 cells; however, only four of them (66.6%) were invasive. Thus, these results suggest that the presence of tpeL gene or TpeL toxin might be required for the adherence of bacteria to HEp-2 cells; however, it could not have any role in the invasion process.

Pathogenicity and genetic profile of oral Porphyromonas species from canine periodontitis.

OBJECTIVE: In this study, the presence of the prtC and fimA genes involved in the pathogenicity of oral Porphyromonas spp. isolated from dogs with periodontitis and healthy, as well as their genetic diversity was investigated. DESIGN: Thirty-two Beagle dogs, 24 with periodontitis and 8 healthy were evaluated. Subgingival samples from only one gingival site of both groups were collected. Bacteria grown in anaerobiosis were identified by RAPID ID 32A kits. From each strain the respective DNA was obtained and used to genotyping by conventional PCR and AP-PCR. RESULTS: Dogs with periodontitis harbored 28 P. gulae, 2 P. creviocaricanis, 1 P. cangingivalis and 7 P. macacae; and from healthy dogs, 11 P. gulae and 5 P. circumdentaria. In P. gulae isolated from periodontal dogs the gene prtC was observed in 19 (67.85%) and in 7 (63.63%) from healthy dogs. P. gulae strains from periodontal dogs harbored either the gene fimA I or fimA II; while strains from healthy dogs harbored the gene fimA I, fimA II, fimA III or fimA IV, as well as 1 P. circumdentaria the gene fimA II. By AP-PCR strains were grouped in different clusters suggesting heterogeneity of these microorganisms. CONCLUSIONS: The results presented herein inform that Porphyromonas spp. isolated from dogs with and without periodontitis harbored the prtC and fimA genes and it could be a role in the establishment of the infectious process.

Alterations of Intestinal Microbiome by Antibiotic Therapy in Hospitalized Children.

The administration of antimicrobial agents leads to an ecological imbalance of the host-microorganisms relationship, and it causes a rapid and significant reduction in the microbial diversity. The aim of the current study was to evaluate the impact of antibiotic therapy on intestinal microbiota of children between 3 and 12 years of age. The fecal samples were collected from hospitalized children (n = 31) and from healthy untreated children (n = 30). The presence of bacteria and their quantities were assessed by culture-based methods and quantitative polymerase chain reaction (qPCR). By culture method, in the children receiving antibiotics, a low recovery of Bifidobacterium spp. (54.8%), Bacteroides spp./Parabacteroides spp. (54.8%), Clostridium spp. (35.5%), and Escherichia coli (74.2%) was observed compared with the children without antibiotic therapy (100%, 80%, 63.3%, and 86.6%, respectively). By qPCR, the children receiving antibiotics showed a lower copy number for all microorganisms, except to Lactobacillus spp. (p = 0.0092). In comparison to the nontreated children, the antibiotic-treated children showed a significantly lower copy number of Bifidobacterium spp. (p = 0.0002), Clostridium perfringens (p < 0.0001), E. coli (p = 0.0268), Methanobrevibacter smithii (p = 0.0444), and phylum Firmicutes (p = 0.0009). In conclusion, our results obtained through qualitative and quantitative analyses, demonstrate that antibiotic therapy affect the intestinal microbiome of children.

Effects of subinhibitory concentrations of clindamycin on the morphological, biochemical and genetic characteristics of Bacteroides fragilis.

The effects of subinhibitory concentrations of clindamycin on the morphological, biochemical and genetic characteristics of species of the Bacteroides fragilis group isolated from children with diarrhea were determined. The minimal inhibitory and subinhibitory concentrations for clindamycin were determined. Minimal inhibitory concentration values ranging from 0.25 to 512 microg mL(-1) were observed. Cultures grown with clindamycin were used to determine the macroscopic morphological characteristics, cellular viability, ultrastructural characteristics and DNA integrity. Clindamycin did not alter colonial morphology, but after 6 h elongated cells were observed. Also, extracellular vesicles and electron-lucent areas inside the cytoplasm were observed. Bacteria treated with clindamycin also showed fragmentation of DNA as determined by electrophoresis. The alterations produced by clindamycin might be indicative of a possible modification of the structures involved in bacterial pathogenesis.

Genetic diversity of oral Fusobacterium nucleatum isolated from patients with different clinical conditions.

The genetic diversity of 23 oral Fusobacterium nucleatum isolated from 15 periodontal patients, eight from seven healthy subjects, nine from nine AIDS patients and two from two Cebus apella monkeys were analyzed. EcoRI restricted the bacterial DNA and 28 ribotypes grouped from A to J groups were obtained. Isolates formed 24 ribotypes which were contained into A, B, C, D, E and F groups, and three reference strains and two clinical isolates of A. actinomycetemcomitans, and E. coli CDC formed four different ribotypes into the G, H, I and J groups. Moreover, from nine F. nucleatum from AIDS patients, six were ribotyped as group C and three as group D. By using ribotyping we distinguished F. nucleatum recovered from different sources. It is possible that isolates from AIDS patients may contain some phenotypic or genotypic factor did not observed in this study.

Role of NOD2 and RIP2 in host-microbe interactions with Gram-negative bacteria: insights from the periodontal disease model.

NOD2 is a member of the NLR family of proteins that participate in the activation of the innate immune response. RIP2 is a downstream kinase activated by both NOD1 and NOD2. There is scarcity of information regarding the relevance of NOD2 in periodontitis, a chronic inflammatory condition characterized by inflammatory bone resorption. We used NOD2-KO and RIP2-KO mice in a model of microbial-induced periodontitis. Heat-killed Aggregatibacter actinomycetemcomitans was injected in the gingival tissues three times/wk for 4 wk. Bone resorption was assessed by µCT analysis; osteoclasts were identified by immunohistochemical staining for TRAP and inflammation was assessed using a severity score system in H/E-stained sections. In vitro studies using primary macrophages assessed the response macrophages using qPCR-based array and multi-ligand ELISA. Bone resorption and osteoclastogenesis were significantly reduced in NOD2-KO mice. Severity of inflammation was not affected. qPCR-focused arrays and multi-ligand ELISA showed that expression of pro-inflammatory mediators was reduced in NOD2- and RIP2-deficient cells. RANKL-induced osteoclastogenesis was impaired in NOD2- and RIP2-deficient macrophages. We conclude that NOD2 is important for osteoclast differentiation and inflammatory bone resorption in vivo and also for the macrophage response to Gram-negative bacteria.

Actinobacillus actinomycetemcomitans-induced periodontal disease in mice: patterns of cytokine, chemokine, and chemokine receptor expression and leukocyte migration.

Although the pathogenesis of periodontal disease (PD) is not well known, cytokines, chemotactic factors and inflammatory cells are certainly involved in the disease outcome. Here, we characterized the evolution of the PD induced by Actinobacillus actinomycetemcomitans in mice, showing that oral inoculation of these bacteria leads to the migration of leukocytes to periodontal tissues and marked alveolar bone resorption. We found the expression of pro-inflammatory and Th1-type cytokines including TNF-alpha, IFN-gamma and IL-12 in periodontal tissues after infection with A. actinomycetemcomitans, from the early stages after infection and throughout the course of the disease. Similar kinetics of expression were found for the chemokines CCL5, CCL4, CCL3 and CXCL10 and for the receptors CCR5 and CXCR3, all of them linked to the Th1-type pattern. The expression of the Th2-type mediators IL-10, CCL1 and their receptors CCR4 and CCR8 was detected only after 30 days of infection, determining a time-dependent mixed pattern of polarized immune response. The chemokine expression was correlated with the presence of polymorphonuclear leukocytes, macrophages, CD4 and CD8 lymphocytes, and B cells in the inflammatory infiltrate. Interestingly, during the predominance of the Th1-type response, a sharp increase in the number of inflammatory cells and intense bone loss was seen. By contrast, after the increased expression of Th2-type mediators, the number of inflammatory cells remained constant. Our data demonstrate that mice subjected to oral inoculation of A. actinomycetemcomitans represent a useful model for the study of PD. In addition, our results suggest that expression of cytokines and chemokines can drive the selective recruitment of leukocyte subsets to periodontal tissues, which could determine the stable or progressive nature of the lesion.