Effects of dietary yeast cell wall on faecal bacteria and fermentation products in adult cats.

This study evaluated the effects of increasing concentrations of spray-dried yeast cell wall (YCW) in diets for healthy adult cats on apparent nutrient digestibility and on bacterial composition and fermentation products in the stool. Fourteen cats with an average weight of 4.40 ± 1.05 kg and an average age of 6.2 ± 0.54 years were used and assigned to treatments in an unbalanced randomized block design (by experimental period) with two blocks and three or four cats per diet in each block. Treatments included: control (0% YCW), 0.2% YCW, 0.4% YCW and 0.6% YCW, totalling seven animals per experimental diet. We found that YCW did not affect body weight, nutrient and food intake, faecal production, faecal score, faecal pH or urine output (p > .05). Regarding faecal bacteria, we observed a linear reduction in Clostridium perfringens, a quadratic reduction in Escherichia coli, and linear increases in Bifidobacterium spp. and Lactobacillus spp. (p < .05) with the inclusion of YCW. Regarding the faecal short-chain fatty acid profile, butyrate, valerate, total biogenic amines, putrescine, cadaverine and histamine increased linearly (p < .05) with the inclusion of YCW. It was concluded that in healthy adult cats, consumption of YCW modulates the faecal bacterial populations, with an increased presence of beneficial bacteria and a reduction in some potentially pathogenic bacteria. It was concluded that YCW modulated the levels of fermentation products. There was an increase in fermentation products coming from carbohydrate metabolism, an important effect that can potentially benefit the intestinal health of cats. The consumption of YCW also increased the fermentation of nitrogen compounds, which have not yet been defined as deleterious or beneficial. The fermentability of carbohydrates and nitrogen compounds may be associated. Therefore, YCW may cause rapid fermentation of both classes of compounds by enhancing the fermentability of one class.

Microbial analysis of root canal and periradicular lesion associated to teeth with endodontic failure.

The quantification of ten microorganisms at the root ends and in the surrounding periradicular lesions was performed. Thirty 3 mm samples root ends and 30 samples of the surrounding chronic periapical infection were collected during apical microsurgery. Samples were triturated, and the bacterial DNA was obtained. The bacterial quantification was performed by using the SYBR Green system. At least one microorganism was detected in all patients. In both the root end and periapical samples, Fusobacterium nucleatum (71.6%), Dialister pneumosintes (58.3%) and Tannerella forsythia (48.3%) were the most prevalent species. Dialister pneumosintes showed statistically significant values in the root end, and F. nucleatum was also significant in the apical periodontitis samples. A statistically significant association between T. forsythia and Porphyromonas gingivalis in the root ends was observed. Bacterial associations from 2 to 7 species were observed in most samples. Extra-radicular and/or intra-radicular infections were present in all teeth with failed endodontic treatment, and showed polymicrobial infection in most cases, with a predominance of F. nucleatum, D. pneumosintes and T. forsythia. When present, Enterococcus faecalis was never found to be the most prevalent species. The presence of a microbial diversity in post-treatment apical periodontitis confirms the polymicrobial and synergistic characteristic of this process. Our results show that the bacterial array associated with the 3 mm root ends and periradicular lesions in post-treatment apical periodontitis are complex and with a high inter-individual variability. These results might be useful to delineate treatment strategies for microbial elimination in apical periodontitis. Further studies are necessary to elucidate the role of these microorganisms in endodontic treatment failures.

Multilocus sequence typing analyses of Clostridium perfringens type A strains harboring tpeL and netB genes.

Clostridium perfringens is an anaerobic bacterium ubiquitous in various environments, especially in soil and the gastrointestinal tract of healthy humans and animals. In this study, multilocus sequence typing protocol was used to investigate genotypic relationships among 40 C. perfringens strains isolated from humans and broiler chicken with necrotic enteritis [NE]. The results indicated a few clonal populations, mainly observed in human strains, with 32.5% of all strains associated with one of three clonal complexes and 30 sequences types. The CC-1 cluster showed an interesting and unexpected result because it contained seven strains [six from animals and one of human origin]. Detection assays for toxin genes tpeL and netB were also performed. The netB gene was only observed in 7.5% of the strains from healthy human. The toxin gene tpeL was detected in 22.5% of the C. perfringens strains isolated from three individuals and in six broilers with NE. Our study describes the role of some C. perfringens strains of human origin acting as reservoirs of virulence genes and sources of infection. In addition, the strains of human and animal origin were found to be genetically distinct but phylogenetically close, and the human strains showed more diversity than the animal strains.

Bacterial short-chain fatty acid metabolites modulate the inflammatory response against infectious bacteria.

Short-chain fatty acids (SCFAs), predominantly acetic, propionic, and butyric acids, are bacterial metabolites with an important role in the maintenance of homeostasis due to their metabolic and immunomodulatory actions. Some evidence suggests that they may also be relevant during infections. Therefore, we aimed to investigate the effects of SCFAs in the effector functions of neutrophils to an opportunistic pathogenic bacterium, Aggregatibacter actinomycetemcomitans. Using a subcutaneous model to generate a mono, isolated infection of A. actinomycetemcomitans, we demonstrated that the presence of the SCFAs in situ did not affect leukocyte accumulation but altered the effector mechanisms of migrating neutrophils by downregulating the production of cytokines, their phagocytic capacity, and killing the bacteria, thus impairing the containment of A. actinomycetemcomitans. Similar effects were observed with bacteria-stimulated neutrophils incubated with SCFAs in vitro. These effects were independent of free-fatty acid receptor 2 (FFAR2) activation, the main SCFA receptor expressed on neutrophils, occurring possibly through inhibition of histone deacetylases because similar effects were obtained by using histone deacetylase inhibitors, such as SAHA, MS-275, and RGFP 966. Considering the findings of this study, we hypothesized that in an infectious condition, SCFAs may exert a detrimental effect on the host by inhibiting neutrophil's effector functions.

Multilocus sequence typing analyses of Clostridium perfringens type A strains harboring tpeL and netB genes

Clostridium perfringens is an anaerobic bacterium ubiquitous in various environments, especially in soil and the gastrointestinal tract of healthy humans and animals. In this study, multilocus sequence typing protocol was used to investigate genotypic relationships among 40 C. perfringens strains isolated from humans and broiler chicken with necrotic enteritis [NE]. The results indicated a few clonal populations, mainly observed in human strains, with 32.5% of all strains associated with one of three clonal complexes and 30 sequences types. The CC-1 cluster showed an interesting and unexpected result because it contained seven strains [six from animals and one of human origin]. Detection assays for toxin genes tpeL and netB were also performed. The netB gene was only observed in 7.5% of the strains from healthy human. The toxin gene tpeL was detected in 22.5% of the C perfringens strains isolated from three individuals and in six broilers with NE. Our study describes the role of some C perfringens strains of human origin acting as reservoirs of virulence genes and sources of infection. In addition, the strains of human and animal origin were found to be genetically distinct but phylogenetically close, and the human strains showed more diversity than the animal strains.

Correlation between body mass index and faecal microbiota from children.

Childhood obesity is an increasing problem at the global level and considered as a risk factor for obesity development and the associated co-morbidities in adult life. In this study, the occurrence of Bacteroides fragilis group, Clostridium spp., Bifidobacterium spp. and Escherichia coli in 84 faecal samples from 30 obese, 24 overweight and 30 lean children was verified by culture technique and quantitative determination by quantitative PCR. In addition, Lactobacillus spp. and Methanobrevibacter smithii were also analysed. A correlation between the body mass index (BMI) and these bacteria was sought. Bacteroides vulgatus, Clostridium perfringens and Bifidobacterium adolescentis were most prevalent in all samples evaluated by culture-method. The B. fragilis group were found at high concentrations in obese and overweight children when compared with the lean ones (p 0.015). The obese and overweight children harboured higher numbers of Lactobacillus spp. than lean children (p 0.022). The faecal concentrations of the B. fragilis group (r = 0.24; p 0.026) and Lactobacillus spp. (r = 0.44; p 0.002) were positively correlated with BMI. Bifidobacterium spp. were found in higher numbers in the lean group than the overweight and obese ones (p 0.042). Furthermore, a negative correlation between BMI and Bifidobacterium spp. copy number (r = -0.22; p 0.039) was observed. Our findings show some difference in the intestinal microbial ecosystem of obese children compared with the lean ones and a significant association between number of Lactobacillus spp. and B. fragilis group and BMI.

CCR5 mediates pro-osteoclastic and osteoclastogenic leukocyte chemoattraction.

Periodontal disease (PD) progression involves the selective leukocyte infiltration into periodontium, supposedly mediated by the chemokine/chemokine receptor system. In this study, we investigated the role of chemokine receptor CCR5 in the immunoregulation of experimental PD in C57BL/6 (WT) and CCR5KO mice. Aggregatibacter actinomycetem comitans infection triggered the chemoattraction of distinct CCR5+ leukocyte subpopulations (determined by flow cytometry): CCR5+F4/80+ leukocytes, which co-express CD14 , CCR2, TNF-α, and IL-1ß, indicative of activated macrophages; and CCR5+CD4+ cells, which co-express CXCR3, IFN-γ, and RANKL, indicative of Th1 lymphocytes, therefore comprising pro-osteoclastic and osteoclastogenic cell subsets, respectively. CCR5KO mice presented a lower PD severity (lower inflammation and alveolar bone loss) when compared with the WT strain, since the migration of F4/80+, TNF-α+, CD4+, and RANKL+ cells specifically decreased due to the lack of CCR5. Also, ELISA analysis demonstrated that the production of TNF-α, IL-1ß, IL-6, IFN-γ, and RANKL in periodontal tissues was significantly decreased in the CCR5KO strain. The periodontal bacterial load and antimicrobial patterns were unaltered in CCR5KO mice. Our results demonstrate that the chemokine receptor is involved in the migration of distinct leukocyte subpopulations throughout experimental PD, being a potential target for therapeutic intervention in PD.

Quantitative detection of enterotoxigenic Bacteroides fragilis subtypes isolated from children with and without diarrhea.

A rapid real-time PCR (RT-PCR) approach was developed to detect the bft gene subtypes in Bacteroides fragilis isolated from fecal samples. DNA obtained from diarrhea (110) and nondiarrhea (150) samples was evaluated. Subtype 1 was observed in 9 (8.2%) diarrhea and 7 (4.7%) nondiarrhea samples. Subtype 2 was not detected in any DNA samples, and subtype 3 was observed in only 1 diarrhea sample. The presence of the bft-1 gene did not show any statistically significant differences between the groups of children. This technique could be used to evaluate a possible correlation between disease and the presence of B. fragilis enterotoxin.

Periodontitis and arthritis interaction in mice involves a shared hyper-inflammatory genotype and functional immunological interferences.

Periodontitis (PD) and rheumatoid arthritis (RA) have been found to be clinically associated and to share the chronic nature of the inflammatory reaction associated with bone resorption activity. However, the mechanisms underlying such association are unknown. Therefore, we examined the basis of Actinobacillus actinomycetemcomitans- and Porphyromonas gingivalis-induced PD and pristane-induced arthritis (PIA) interaction in mice. Higher severity PD in the genetically inflammation prone acute inflammatory reactivity maximum (AIRmax) mice strain was associated with higher levels of TNF-alpha, IL-1beta, IL-17, matrix metalloproteinase (MMP)-13, and RANKL, whereas PD/PIA co-induction resulted in even higher levels of IL-1beta, IFN-gamma, IL-17, RANKL, and MMP-13 levels. Conversely, PD/PIA co-induction in AIRmin strain did not alter the course of both pathologies. PIA/PD co-induction resulted in altered expression of T-cell subsets transcription factors expression, with T-bet and RORgamma levels being upregulated, whereas GATA-3 levels were unaltered. Interestingly, PIA induction resulted in alveolar bone loss, such response being highly dependent on the presence of commensal oral bacteria. No differences were found in PIA severity parameters by PD co-induction. Our results show that the interaction between experimental PD and arthritis in mice involves a shared hyper-inflammatory genotype and functional interferences in innate and adaptive immune responses.

Periodontitis and arthritis interaction in mice involves a shared hyper-inflammatory genotype and functional immunological interferences

Periodontitis (PD) and rheumatoid arthritis (RA) have been found to be clinically associated and to share the chronic nature of the inflammatory reaction associated with bone resorption activity. However, the mechanisms underlying such association areunknown. Therefore, we examined the basis of Actinobacillus actinomycetemcomitans- and Porphyromonas gingivalis-inducedPD and pristane-induced arthritis (PIA) interaction in mice. Higher severity PD in the genetically inflammation prone acute inflammatory reactivity maximum (AIRmax) mice strain was associated with higher levels of TNF-a, IL-1b, IL-17, matrix metalloproteinase (MMP)-13, and RANKL, whereas PD/PIA co-induction resulted in even higher levels of IL-1b, IFN-g, IL-17, RANKL, and MMP-13 levels. Conversely, PD/PIA co-induction in AIRmin strain did not alter the course of both pathologies. PIA/PD co-induction resulted in altered expression of T-cell subsets transcription factors expression, with T-bet and RORg levels being upregulated, whereas GATA-3 levels were unaltered. Interestingly, PIA induction resulted in alveolar bone loss, such response being highly dependent on the presence of commensal oral bacteria. No differences were found in PIA severity parameters by PD co-induction. Our results show that the interaction between experimental PD and arthritis in mice involves a shared hyper-inflammatory genotype and functional interferences in innate and adaptive immune responses.

Experimental periodontitis in mice selected for maximal or minimal inflammatory reactions: increased inflammatory immune responsiveness drives increased alveolar bone loss without enhancing the control of periodontal infection.

BACKGROUND AND OBJECTIVE: Inflammatory immune reactions that occur in response to periodontopathogens are thought to protect the host against infection, but may trigger periodontal destruction. However, the molecular and genetic mechanisms underlying host susceptibility to periodontal infection and to periodontitis development have still not been established in detail. MATERIAL AND METHODS: In this study, we examined the mechanisms that modulate the outcome of Aggregatibacter (Actinobacillus) actinomycetemcomitans-induced periodontal disease in mice mouse strains selected for maximal (AIRmax) or minimal (AIRmin) inflammatory reactions. RESULTS: Our results showed that AIRmax mice developed a more severe periodontitis than AIRmin mice in response to A. actinomycetemcomitans infection, and this periodontitis was characterized by increased alveolar bone loss and inflammatory cell migration to periodontal tissues. In addition, enzyme-linked immunosorbent assays demonstrated that the levels of the cytokines interleukin-1beta, tumor necrosis factor-alpha and interleukin-17 were higher in AIRmax mice, as were the levels of matrix metalloproteinase (MMP)-2, MMP-13 and receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA levels. However, the more intense inflammatory immune reaction raised by the AIRmax strain, in spite of the higher levels of antimicrobial mediators myeloperoxidase and inducible nitric oxide synthase, did not enhance the protective immunity to A. actinomycetemcomitans infection, because both AIRmax and AIRmin strains presented similar bacterial loads in periodontal tissues. In addition, the AIRmax strain presented a trend towards higher levels of serum C-reactive protein during the course of disease. CONCLUSION: Our results demonstrate that the intensity of the inflammatory immune reaction is associated with the severity of experimental periodontitis, but not with the control of A. actinomycetemcomitans periodontal infection, suggesting that the occurrence of hyperinflammatory genotypes may not be an evolutionary advantage in the complex host-pathogen interaction observed in periodontal diseases.

Tumor necrosis factor-alpha -308G/A single nucleotide polymorphism and red-complex periodontopathogens are independently associated with increased levels of tumor necrosis factor-alpha in diseased periodontal tissues.

BACKGROUND AND OBJECTIVE: Inflammatory cytokines such as tumor necrosis factor-alpha are involved in the pathogenesis of periodontal diseases. A high between-subject variation in the level of tumor necrosis factor-alpha mRNA has been verified, which may be a result of genetic polymorphisms and/or the presence of periodontopathogens such as Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola (called the red complex) and Aggregatibacter actinomycetemcomitans. In this study, we investigated the effect of the tumor necrosis factor-alpha (TNFA) -308G/A gene polymorphism and of periodontopathogens on the tumor necrosis factor-alpha levels in the periodontal tissues of nonsmoking patients with chronic periodontitis (n = 127) and in control subjects (n = 177). MATERIAL AND METHODS: The TNFA -308G/A single nucleotide polymorphism was investigated using polymerase chain reaction-restriction fragment length polymorphism analysis, whereas the tumor necrosis factor-alpha levels and the periodontopathogen load were determined using real-time polymerase chain reaction. RESULTS: No statistically significant differences were found in the frequency of the TNFA -308 single nucleotide polymorphism in control and chronic periodontitis groups, in spite of the higher frequency of the A allele in the chronic periodontitis group. The concomitant analyses of genotypes and periodontopathogens demonstrated that TNFA -308 GA/AA genotypes and the red-complex periodontopathogens were independently associated with increased levels of tumor necrosis factor-alpha in periodontal tissues, and no additive effect was seen when both factors were present. P. gingivalis, T. forsythia and T. denticola counts were positively correlated with the level of tumor necrosis factor-alpha. TNFA -308 genotypes were not associated with the periodontopathogen detection odds or with the bacterial load. CONCLUSION: Our results demonstrate that the TNFA -308 A allele and red-complex periodontopathogens are independently associated with increased levels of tumor necrosis factor-alpha in diseased tissues of nonsmoking chronic periodontitis patients and consequently are potentially involved in determining the disease outcome.

iNOS-derived nitric oxide modulates infection-stimulated bone loss.

Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) plays an important role in host defense, as well as in inflammation-induced tissue lesions. Here we evaluated the role of NO in bone loss in bacterial infection-induced apical periodontitis by using iNOS-deficient mice (iNOS(-/-)). The iNOS(-/-) mice developed greater inflammatory cell recruitment and osteolytic lesions than WT mice. Moreover, tartrate-resistant acid-phosphatase-positive (TRAP(+)) osteoclasts were significantly more numerous in iNOS(-/-) mice. Furthermore, the increased bone resorption in iNOS(-/-) mice also correlated with the increased expression of receptor activator NF-kappaB (RANK), stromal-cell-derived factor-1 alpha (SDF-1 alpha/CXCL12), and reduced expression of osteoprotegerin (OPG). These results show that NO deficiency was associated with an imbalance of bone-resorption-modulating factors, leading to severe infection-stimulated bone loss.

Adherence and invasion of Bacteroidales isolated from the human intestinal tract.

Members of the genera Bacteroides and Parabacteroides are important constituents of both human and animal intestinal microbiota, and are significant facultative pathogens. In this study, the ability of Bacteroides spp. and Parabacteroides distasonis isolated from both diarrhoeal and normal stools (n = 114) to adhere to and invade HEp-2 cells was evaluated. The presence of putative virulence factors such as capsule and fimbriae was also investigated. Adherence to HEp-2 cells was observed in 75.4% of the strains, which displayed non-localized clusters. Invasion was observed in 37.5% and 26% of the strains isolated from diarrhoeal and non-diarrhoeal stools, respectively. All strains displayed a capsule, whereas none of them showed fimbriae-like structures. This is the first report of the ability of Bacteroides spp. and P. distasonis to adhere to and invade cultured HEp-2 epithelial cells.

Antagonic effect of the inhibition of inducible nitric oxide on the mortality of mice acutely infected with Escherichia coli and Bacteroides fragilis.

Sepsis, the leading cause of death in intensive care units, is associated with overproduction of nitric oxide (NO) due to inducible NO synthase (iNOS), responsible for some of the pathologic changes. Aminoguanidine (AG) is a selective iNOS inhibitor with reported inconsistent actions in sepsis. To investigate the influence of iNOS, we studied models of acute bacterial sepsis using acute challenges with aerobic (Escherichia coli) and anaerobic (Bacteroides fragilis) bacteria in the presence of AG. Six-week-old, 23 g, male and female BALB/c and C57Bl/6j mice, in equal proportions, were inoculated (ip) with bacteria in groups of 4 animals for each dose and each experiment in the absence or presence of AG (50 mg/kg, ip, starting 24 h before challenge and daily until day 6) and serum nitrate was measured by chemiluminescence. Both types of bacteria were lethal to mice, with an LD50 of 6 nephelometric units (U) for E. coli and 8 U for B. fragilis. Nitrate production peaked on the second day after E. coli inoculation with 8 and 6 U (P < 0.05), but was absent after non-lethal lower doses. After challenge with B. fragilis this early peak occurred at all tested doses after 24 h, including non-lethal ones (P < 0.05). AG-treated mice challenged with E. coli presented higher survival (P < 0.05) and increased LD50. AG-treated mice challenged with B. fragilis had lower LD50 and higher mortality. Control AG-treated animals presented no toxic effects. The opposite effect of iNOS blockade by AG in these models could be explained by restriction of oxygen for immune cells or an efficient action of NO in anaerobic localized infections. The antagonic role of NO production observed in our bacterial models could explain the reported discrepancy of NO action in sepsis.

Antagonic effect of the inhibition of inducible nitric oxide on the mortality of mice acutely infected with Escherichia coli and Bacteroides fragilis

Sepsis, the leading cause of death in intensive care units, is associated with overproduction of nitric oxide (NO) due to inducible NO synthase (iNOS), responsible for some of the pathologic changes. Aminoguanidine (AG) is a selective iNOS inhibitor with reported inconsistent actions in sepsis. To investigate the influence of iNOS, we studied models of acute bacterial sepsis using acute challenges with aerobic (Escherichia coli) and anaerobic (Bacteroides fragilis) bacteria in the presence of AG. Six-week-old, 23 g, male and female BALB/c and C57Bl/6j mice, in equal proportions, were inoculated (ip) with bacteria in groups of 4 animals for each dose and each experiment in the absence or presence of AG (50 mg/kg, ip, starting 24 h before challenge and daily until day 6) and serum nitrate was measured by chemiluminescence. Both types of bacteria were lethal to mice, with an LD50 of 6 nephelometric units (U) for E. coli and 8 U for B. fragilis. Nitrate production peaked on the second day after E. coli inoculation with 8 and 6 U (P < 0.05), but was absent after non-lethal lower doses. After challenge with B. fragilis this early peak occurred at all tested doses after 24 h, including non-lethal ones (P < 0.05). AG-treated mice challenged with E. coli presented higher survival (P < 0.05) and increased LD50. AG-treated mice challenged with B. fragilis had lower LD50 and higher mortality. Control AG-treated animals presented no toxic effects. The opposite effect of iNOS blockade by AG in these models could be explained by restriction of oxygen for immune cells or an efficient action of NO in anaerobic localized infections. The antagonic role of NO production observed in our bacterial models could explain the reported discrepancy of NO action in sepsis.

The dual role of p55 tumour necrosis factor-alpha receptor in Actinobacillus actinomycetemcomitans-induced experimental periodontitis: host protection and tissue destruction.

Inflammatory immune reactions in response to periodontopathogens are thought to protect the host against infection, but may trigger periodontal destruction. Thus, we examined the mechanisms by which the proinflammatory cytokine tumour necrosis factor (TNF)-alpha modulates the outcome of Actinobacillus actinomycetemcomitans-induced periodontal disease in mice. Our results showed that TNF-alpha receptor p55-deficient mice [p55TNF-knock-out (KO)] developed a less severe periodontitis in response to A. actinomycetemcomitans infection, characterized by significantly less alveolar bone loss and inflammatory reaction. Real-time polymerase chain reaction (PCR) demonstrated that levels of chemokines (CXCL1, 3 and 10; CCL3 and 5) and their receptors (CXCR2 and 3, CCR5) were lower in p55TNF-KO mice, as were matrix metalloproteinase (MMP)-1, 2 and 9 and receptor activator of nuclear factor kB ligand (RANKL) mRNA levels. However, the absence of the TNF-alpha p55 results in an impairment of protective immunity to A. actinomycetemcomitans infection, characterized by increased bacterial load and higher levels of C-reactive protein during the course of disease. Such impaired host response may be the result of the reduced chemoattraction of lymphocytes, neutrophils and macrophages, and reduced inducible nitric oxide synthase expression (iNOS) and myeloperoxidase (MPO) production in periodontal tissues of p55 TNF-KO mice. Our results demonstrate the mechanisms involved determining periodontal disease severity by TNF-alpha receptor p55, and its role in providing immune protection to A. actinomycetemcomitans periodontal infection.

Plasmid-related resistance to cefoxitin in species of the Bacteroides fragilis group isolated from intestinal tracts of calves.

Species of the Bacteroides fragilis group are considered the most common anaerobe in human and animal infections and also harbor plasmids conferring resistance to several antibiotics. In this study, resistance to cefoxitin, plasmid profile and beta-lactamase production in species of the B. fragilis group isolated from intestinal tracts of calves were evaluated. One hundred sixty-one B. fragilis group bacteria isolated from calves with and without diarrhea were analyzed. Cefoxitin susceptibility was performed using an agar dilution method, beta-lactamase production by using a nitrocefin method, and plasmid extraction by using a commercial kit. Minimal inhibitory concentration values for cefoxitin ranged from 32 to > 512 microg/ml, and 47 bacteria (29.2%) were resistant to cefoxitin (breakpoint 16 microl). Only seven isolates harbored plasmids varying from 6.0 to 5.0 kb, and a 5.5-kb plasmid in B. vulgatus Bd26e and B. fragilis Bc5j might be related to cefoxitin resistance. beta-lactamase was detected in 33 (70.2%) isolates. The cepA gene was observed in total DNA and in the 5.5-kb plasmid. The plasmid presence in organisms isolated from cattle may be important in ecologic terms, and it needs further study.

Cytokine pattern determines the progression of experimental periodontal disease induced by Actinobacillus actinomycetemcomitans through the modulation of MMPs, RANKL, and their physiological inhibitors.

OBJECTIVE: Inflammatory and immune reactions raised in response to periodontopathogens are thought to trigger periodontal tissue destruction. We therefore investigated the expression of matrix metalloproteinases (MMPs) and the osteoclastogenic factor RANKL (receptor activator of nuclear factor-kappaB ligand), their respective inhibitors TIMPs (tissue inhibitors of metalloproteinases) and OPG (osteoprotegerin) and their possible correlation with the expression of inflammatory and regulatory cytokines in the course of experimental periodontal disease in mice. METHODS: We characterized the time course of leukocyte migration and alveolar bone loss in C57BL/6 mice infected with Actinobacillus actinomycetemcomitans. Quantitative polymerase chain reaction (RealTime PCR) and ELISA were performed to determine the expression of MMPs, TIMPs, RANKL, OPG and cathepsin K, interleukin-1beta, tumor necrosis factor-alpha, interferon-gamma, interleukin-12, interleukin-4 and interleukin-10 in periodontal tissue samples harvested throughout the course of experimental disease. RESULTS: Oral inoculation of A. actinomycetemcomitans results in an intense and widespread migration of leukocytes to the gingival tissues, besides marked alveolar bone resorption. Our data also demonstrate two distinct patterns of MMP/TIMP and RANKL/OPG expression in the course of experimental periodontal disease. The expression of MMPs (MMP-1, 2 and 9) and RANKL was correlated with the expression of interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma, in a time period characterized by the intense increase of inflammatory reaction and alveolar bone loss. On the other hand, interleukin-4 and interleukin-10 were associated with higher expression of TIMPs (TIMP 1, 2 and 3) and OPG, with a lower expression of MMPs and RANKL, and with reduced rates of increase of cellular infiltration in periodontal tissues and alveolar bone loss. CONCLUSIONS: It is possible that the pattern of cytokines produced in periodontal tissues determines the progression and the severity of experimental periodontal disease, controlling the breakdown of soft and bone tissues through the balance between MMPs/TIMP and RANKL/OPG expression in gingival tissues.

Adherence of Actinobacillus actinomycetemcomitans on oral epithelial cells.

Actinobacillus actinomycetemcomitans is capable of colonizing mucosa and dental plaque and plays an important role in periodontal disease in young peoples and adult. Adherence mechanisms on epithelial cells, tooth or oral bacteria and gingival invasion probably are the initial steps in the pathogenesis of gingivitis or periodontitis. In this study, the adherence of A. actinomycetemcomitans on oral epithelial cells following subculturing were examined. The adherence on oral epithelial cells showed high in all the isolates values but with differences among them and at each time of subculturing. The adherence of A. actinomycetemcomitans FDC Y4 was stable in each of the subcultures. However, adhesion values of all the tested isolates were different except for strains #1, #38 and Y4, suggesting a heterogenicity within this microbial group. Morphologic variations were observed in extracellular structures of the A. actinomycetemcomitans tested. The adhesion process on oral epithelial cells of this organism can be influenced by subcultures, but additional studies are necessary to verify the influence of subculturing on adherence or other virulence factors.