Perillyl alcohol has antibacterial effects and reduces ROS production in macrophages

Abstract Natural products have emerged as a rich source of bioactive compounds for adjunctive treatments of many infectious and inflammatory conditions, including periodontitis. Among the monoterpenes with significant biological properties, there is the perillyl alcohol (POH), which can be found in several essential oils and has shown immunomodulatory properties in recent studies, which may be interesting in the treatment of non-neoplastic inflammatory disorders. Objective To determine the antibacterial and immune modulatory activities of the POH. Methodology The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the POH for two significant Gram-negative periodontal pathogens were determined by macrodilution and subculture, respectively. Cell proliferation and cytotoxicity in RAW 264.7 macrophages were determined by Trypan Blue and mitochondrial enzymatic activity assay. The modulation of reactive oxygen species (ROS) was analyzed by flow cytometry and expression of TNF and arginase-1 by real-time PCR. Results The POH was effective against P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) with MIC= MBC=1600 μM. No cytotoxicity up to 100 µM was observed on macrophages. The cell proliferation was inhibited from 48 hours at 100 μM (p<0.05) and 250 μM (p<0.01). The POH increased ROS production at both 10 μM and 100 μM (p<0.05) in unstimulated cells. The PMA-induced ROS production was not affected by POH, whereas 100 μM significantly reduced lipopolysaccharide-induced (LPS-induced) ROS. The expression of TNF was not affected by POH in unstimulated cells or in cells polarized to M1 phenotype, whereas both concentrations of POH reduced (p<0.05) the expression of arginase-1 in M2-polarized macrophages. Conclusion The POH has antibacterial activity against periodontal pathogens and reduced proliferation of murine macrophages without significant cytotoxicity at concentrations up to 100 μM. In addition, the POH reduced the LPS-induced ROS and the expression of arginase-1 in M2-polarized macrophages.

Aggregatibacter actinomycetemcomitans y Fusobacterium nucleatum en biopelículas subgingivales de pacientes brasileños con y sin enfermedad periodontal: comparación de dos métodos de detección / Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum in subgingival biofilms from Brazilian patients with and without periodontal disease: comparison of two detection methods

Objetivo: Realizar la detección comparativa de cepas de A. actinomycetemcomitans y F. nucleatum de muestras subgingivales por los métodos de cultivo y de reacción en cadena de la polimerasa (PCR). Métodos: Fueron evaluados 50 pacientes con periodontitis crónica (P) y 50 pacientes sanos (S). Las muestras fueron colectadas de bolsas periodontales y surcos gingivales. El cultivo bacteriano fue realizado en agar tripticasa de soya-suero de caballo-bacitracina-vancomicina, e incubado en anaerobiosis. La identificación bac-teriana fue por métodos bioquímicos de fermentación de carbohidratos y por PCR. Resultados: Por el método de cultivo, de las 50 muestras de periodontitis, 9 (18%) fueron positivas para A. actinomycetemcomitans aislándose 17 cepas. También, de esas muestras, 10 (20%) fueron positivas para F. nucleatum aislándose 19 cepas. De las 50 muestras de pacientes sanos, solamente 1 (2%) fue positiva para A. actinomycetemcomitans obteniéndose 2 cepas, y 12 (24%) positivas para F. nucleatum con 18 cepas. Por PCR fueron observadas diferencias en la detección de A. actinomycetemcomitans, entre los tres pares de partidores utilizados, para muestras de bolsa periodontal y surco gingival: partidor AA, 96% y 86%; partidor FU, 48% y 42%; y partidor ASH, 24% y 6%. Los porcentajes de detección para F. nucleatum de muestras de P y S fueron: partidor FN-5047, 36% y 18%; y partidor 505-S, 8% para ambas muestras colectadas. Cepas de A. actinomycetemcomitans biotipo II fueron las más prevalentes. Conclusiones: El método de PCR fue más sensible y específico en la detección bacteriana que el cultivo. Palabras clave: Aggregatibacter actinomycetemcomitans; Fusobacterium nucleatum; Bacterias anaerobias gram-negativas; Periodontitis.

Objective: A comparative detection of strains of A. actinomycetemcomitans and F. nucleatum directly from subgingival samples was performed by culture and polymerase chain reaction (PCR) methods. Methods: Fifty patients with chronic periodontitis (P) and 50 healthy patients (S) were evaluated. Subgingival samples were collected from periodontal pockets and gingival sulcus. Bacterial culture was performed on trypticase soy-horse serum-bacitracin-vancomycin agar and incubated in anaerobiosis. Bacterial identification was done by biochemical methods of carbohydrate fermentation and by PCR. Results:By culture method, of the 50 samples of periodontitis, 9 (18%) were positive for A. actinomycetemcomitans isolating 17 strains. Also, of these samples, 10 (20%) were positive for F. nucleatum isolating 19 strains. Of the 50 samples from healthy patients, only 1 (2%) was positive for A. actinomycetemcomitans, obtaining 2 strains, and 12 (24%) positive for F. nucleatum with 18 strains. Differences were observed in the detection of A. actinomycetemcomitans among the three pairs of primers used, for periodontal pocket and gingival sulcus samples: primer AA, 96% and 86%; primer FU, 48% and 42%; and primer ASH, 24% and 6%. The percentages of detection for F. nucleatum of samples from P and S were: primer FN-5047, 36% and 18%; and primer 505-S, 8% for both samples collected. Strains of A. actinomycetemcomitans biotype II were the most preva-lent. Conclusions: The PCR method was more sensitive and specific in the bacterial detection than the culture. Keywords: Aggregatibacter actinomycetemcomitans; Fusobacterium nucleatum; Gram-negative anaerobic bacteria; Periodontitis.

Enterotoxigenic and non-enterotoxigenic Bacteroides fragilis from fecal microbiota of children

Enterotoxigenic Bacteroides fragilis (ETBF) is an important part of the human and animal intestinal microbiota and is commonly associated with diarrhea. ETBF strains produce an enterotoxin encoded by the bft gene located in the B. fragilis pathogenicity island (BfPAI). Non-enterotoxigenic B. fragilis (NTBF) strains lack the BfPAI and usually show two different genetic patterns, II and III, based on the absence or presence of a BfPAI-flanking region, respectively. The incidence of ETBF and NTBF strains in fecal samples isolated from children without acute diarrhea or any other intestinal disorders was determined. All 84 fecal samples evaluated were B. fragilis-positive by PCR, four of them harbored the bft gene, 27 contained the NTBF pattern III DNA sequence, and 52 were considered to be NTBF pattern II samples. One sample was positive for both ETBF and NTBF pattern III DNA sequences. All 19 B. fragilis strains isolated by the culture method were bft-negative, 9 belonged to pattern III and 10 to pattern II. We present an updated overview of the ETBF and NTBF incidence in the fecal microbiota of children from Sao Paulo City, Brazil.

Association between periodontal condition and subgingival microbiota in women during pregnancy: a longitudinal study

Objectivo In this study, the gingival conditions and the quantitative detection for Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia in pregnant women were determined. Material and Methods Quantitative determinations of periodontal bacteria by using a SyBr green system in women during pregnancy were performed. Women at the 2nd and 3rd trimesters of pregnancy and non-pregnant women were included in this study. A. actinomycetemcomitans was observed in high numbers in women at the 2nd and 3rd trimesters of pregnancy with a significant difference (p<0.05). F. nucleatum and P. intermedia were also observed in high levels. Results and Conclusion Our results show that pregnant women are more susceptible to gingivitis, and the presence of A. actinomycetemcomitans in subgingival biofilm might be taken into account for the treatment of periodontal disease. .

Bacteriological analysis of necrotic pulp and fistulae in primary teeth

Objectives: Primary teeth work as guides for the eruption of permanent dentition, contribute for the development of the jaws, chewing process, preparing food for digestion, and nutrient assimilation. Treatment of pulp necrosis in primary teeth is complex due to anatomical and physiological characteristics and high number of bacterial species present in endodontic infections. The bacterial presence alone or in association in necrotic pulp and fistula samples from primary teeth of boys and girls was evaluated. Material and Methods: Necrotic pulp (103) and fistula (7) samples from deciduous teeth with deep caries of 110 children were evaluated. Bacterial morphotypes and species from all clinical samples were determined. Results: A predominance of gram-positive cocci (81.8%) and gram-negative coccobacilli (49.1%) was observed. In 88 out of 103 pulp samples, a high prevalence of Enterococcus spp. (50%), Porphyromonas gingivalis (49%), Fusobacterium nucleatum (25%) and Prevotella nigrescens (11.4%) was observed. Porphyromonas gingivalis was detected in three out of seven fistula samples, Enterococcus spp. in two out of seven samples, and F. nucleatum, P. nigrescens and D. pneumosintes in one out of seven samples. Conclusions: Our results show that Enterococcus spp. and P. gingivalis were prevalent in necrotic pulp from deciduous teeth in boys from 2 to 5 years old, and that care of the oral cavity of children up to five years of age is important. .

Clostridium perfringens e a enterite necrótica em frangos: principais fatores de virulência, genéticos e moleculares

Clostridium perfringens é o causador da enterite necrótica que afeta a produção de frangos de corte no mundo todo. Essa bactéria produz diversas toxinas e causa lesões no intestino, tendo como consequências a elevada mortalidade e perdas econômicas devido à baixa produtividade. Nesta revisão são apresentados os principais fatores de virulência, a susceptibilidade aos antimicrobianos e a diversidade genética de C. perfringens isolados de frangos com enterite necrótica.

Clostridium perfringens cause necrotic enteritis affecting the poultry production worldwide. This bacterium produces various toxins and causes lesions in the intestine producing high mortality and economic loss due to the low productivity.In this review, the major virulence factors, antimicrobial susceptibility and genetic diversity of C. perfringens from chickens with necrotic enteritis are showed.

Periodontopatógenos e riscos de complicações na gravidez / Periodontopathogens and risks of pregnancy complications. A review of the literature

A doença periodontal é um processo inflamatório crônico dos tecidos periodontais, causada por bactérias gram-negativas anaeróbicas presentes no biofilme dentário. Esse foco infeccioso pode manifestar-se em sítios corporais distantes ou sistêmicos, quando essas bactérias alcançam a corrente sanguínea. Os objetivos desse estudo de revisão são: demonstrar a relação entre doença periodontal e prematuridade, o efeito do tratamento periodontal sobre o nascimento de prematuros, e se microrganismos periodontopatogênicos são capazes de induzir efeitos adversos na gravidez quando os mesmos são encontrados na placenta. Esse estudo constituiu-se de uma revisão da literatura com artigos científicos selecionados através de bancos de dados Scielo, Bireme, Medline e Lilacs, publicados nos anos de 1980 a 2013. Entre os estudos selecionados, todos relacionaram a doença periodontal como sendo um fator de risco importante a ser considerado na gravidez, pois a presença de patógenos orais associou-se à prematuridade, baixo peso fetal ao nascer e infecções perinatais. Portanto, segundo a literatura consultada, a infecção periodontal em mulheres grávidas não deve ser negligenciada pois, se essa doença favorece complicações gestacionais, a atenção à saúde periodontal das gestantes deve estar incluída nas ações de cuidados do pré-natal.

Periodontal disease is a chronic inflammatory process of periodontal tissues caused by gram- negative anaerobic bacteria present in the biofilm. This infection may occur in distant or systemic body sites, when these bacteria reach the bloodstream. The aims of this review study are to demonstrate the relationship between periodontal disease and preterm birth, the effect of the periodontal treatment on preterm birth, and whether periodontopathogenic microorganisms can induce adverse effects on pregnancy when they are found in the placenta. This study is a literature review with scientific papers selected through Scielo Bireme, Medline and Lilacs databases and published between 1980 and 2013. All selected studies considered the periodontal disease as an important risk factor for the pregnancy, since the presence of oral pathogens was associated with premature birth, low birth weight and perinatal infections. Therefore, according to the literature, periodontal infection in pregnant women should not be underestimated, because it favors pregnancy complications, and periodontal health care to pregnant women should be included in the prenatal care.

AP-PCR and Antimicrobial Susceptibility Patterns of Fusobacterium nucleatum Associated with Chronic Periodontitis among Patients at Lagos University Teaching Hospital.

Aims: To isolate, identify and evaluate the genetic diversity and antimicrobial susceptibility of F. nucleatum recovered from Nigerian patients with chronic periodontitis. Study Design: Cross-sectional design. Place and Duration of Study: Department of Medical Microbiology and Parasitology, College of Medicine, University of Lagos, between January 2007 and July 2008. Methodology: We analyzed F. nucleatum species recovered from Nigerian patients with chronic periodontitis. Bacterial identification was done using colonial morphology; Grams stain reaction, conventional biochemical tests, API 20-A and Polymerase chain reaction (PCR). The minimum inhibitory concentration (MIC) of 6 antibiotics was determined by agar dilution method on Brucella blood agar while the bacterial genetic diversity was studied using the Arbitrarily Primed-PCR (AP-PCR) method with the arbitrary primer OPA-05. The interrelationship and genetic similarity matrix among the isolates was determined and by Numerical taxonomy and multivariate analysis system (NTSYS-pc) statistical package. Results: We obtained 48 isolates of F. nucleatum from 50 Nigerian patients (28 males and 22 females) with chronic periodontitis. They were susceptible to metronidazole, clindamycin, cefoxitin, tetracycline, amoxicillin and clavulanate. One was resistant to amoxicillin (MIC >32 μg/ml) and produced β-lactamase. The isolates were further placed into five groups (A, B, C, D and E) based on their AP-PCR profile. Conclusion: The AP-PCR analysis showed heterogeneity among strains. By using APPCR, we observed a single β-lactamase producing clone resistant to amoxicillin which eventually formed a distinct group showing that such genetic difference may have contributed to the formation of a separate clone.

Antimicrobial resistance and prevalence of resistance genes in intestinal Bacteroidales strains

OBJECTIVE: This study examined the antimicrobial resistance profile and the prevalence of resistance genes in Bacteroides spp. and Parabacteroides distasonis strains isolated from children's intestinal microbiota. METHODS: The susceptibility of these bacteria to 10 antimicrobials was determined using an agar dilution method. β-lactamase activity was assessed by hydrolysis of the chromogenic cephalosporin of 114 Bacteriodales strains isolated from the fecal samples of 39 children, and the presence of resistance genes was tested using a PCR assay. RESULTS: All strains were susceptible to imipenem and metronidazole. The following resistance rates were observed: amoxicillin (93 percent), amoxicillin/clavulanic acid (47.3 percent), ampicillin (96.4 percent), cephalexin (99 percent), cefoxitin (23 percent), penicillin (99 percent), clindamycin (34.2 percent) and tetracycline (53.5 percent). P-lactamase production was verified in 92 percent of the evaluated strains. The presence of the cfiA, cepA, ermF, tetQ and nim genes was observed in 62.3 percent, 76.3 percent, 27 percent, 79.8 percent and 7.8 percent of the strains, respectively. CONCLUSIONS: Our results indicate an increase in the resistance to several antibiotics in intestinal Bacteroides spp. and Parabacteroides distasonis and demonstrate that these microorganisms harbor antimicrobial resistance genes that may be transferred to other susceptible intestinal strains.

Microbiota associated with chronic osteomyelitis of the jaws

Chronic osteomyelitis of maxilla and mandible is rare in industrialized countries and its occurrence in developing countries is associated with trauma and surgery, and its microbial etiology has not been studied thoroughly. The aim of this investigation was to evaluate the microbiota associated with osteomyelitis of mandible or maxilla from some Brazilian patients. After clinical and radiographic evaluation, samples of bone sequestra, purulent secretion, and biopsies of granulomatous tissues from twenty-two patients with chronic osteomyelitis of mandible and maxilla were cultivated and submitted for pathogen detection by using a PCR method. Each patient harbored a single lesion. Bacterial isolation was performed on fastidious anaerobe agar supplemented with hemin, menadione and horse blood for anaerobes; and on tryptic soy agar supplemented with yeast extract and horse blood for facultative bacteria and aerobes. Plates were incubated in anaerobiosis and aerobiosis, at 37ºC for 14 and 3 days, respectively. Bacteria were cultivated from twelve patient samples; and genera Actinomyces, Fusobacterium, Parvimonas, and Staphylococcus were the most frequent. By PCR, bacterial DNA was detected from sixteen patient samples. The results suggest that cases of chronic osteomyelitis of the jaws are usually mixed anaerobic infections, reinforcing the concept that osteomyelitis of the jaws are mainly related to microorganisms from the oral environment, and periapical and periodontal infections may act as predisposing factors.

Occurrence of Aggregatibacter actinomycetemcomitans in Brazilian indians from Umutina Reservation, Mato Grosso, Brazil

Aggregatibacter actinomycetemcomitans is associated with periodontal disease, especially localized aggressive periodontitis, produces a potent leukotoxin and its distribution is influenced by ethnic characteristics of the population. Objective: Using culture and polymerase chain reaction (PCR) techniques, this study evaluated the occurrence of this microorganism and the distribution of leukotoxic strains isolated from Indians belonging to the Umutima Reservation, Mato Grosso, Brazil. MATERIAL AND METHODS: Forty-eight native Brazilians with gingivitis and 38 with chronic periodontitis, belonging to Umutina, Paresi, Bororo, Bakairi, Kayabi, Irantxe, Nambikwara and Terena ethnicities, were studied. Subgingival, supragingival and saliva samples of each patient were collected and transferred to VMGA III medium and to ultra pure Milli Q water. Bacteria were grown on TSBV agar and incubated in anaerobiosis (90 percent N2 + 10 percent CO2) at 37ºC for 72 h. The presence of the ltx promoter was determined by PCR, and a 530 bp deletion in the promoter was evaluated by using specific primers. RESULTS: A. actinomycetemcomitans was isolated from 8.33 percent of saliva, supragingival and subgingival samples from patients with gingivitis and from 18.42 percent of saliva and supragingival biofilm, and 26.32 percent subgingival biofilm from patients with chronic periodontitis. By PCR, the bacterial DNA was detected in 8.33 percent of saliva, supragingival and subgingival biofilms from patients with gingivitis and from 23.68 percent of saliva, 28.95 percent supragingival biofilm and 34.21 percent subgingival biofilm from patients with periodontitis. All strains were grouped as non-JP2 clones based on the absence of deletion in the leukotoxin promoter. Differences among the microbial and clinical parameters in patients were analyzed by using the Mann-Whitney, Chi-square or Fisher's exact tests. CONCLUSIONS: The present results suggest that A. actinomycetemcomitans ...

Distribution of biotypes and leukotoxic activity of Aggregatibacter actinomycetemcomitans isolated from Brazilian patients with chronic periodontitis / Distribuição de biótipos e atividade leucotóxica de Aggregatibacter actinomycetemcomitans isolados de pacientes brasileiros com periodontite crônica

Aggregatibacter actinomycetemcomitans is an important etiologic agent of the periodontitis and is associated with extra-oral infections. In this study, the detection of the ltxA gene as well as the ltx promoter region from leukotoxic A. actinomycetemcomitans isolated from 50 Brazilian patients with periodontitis and 50 healthy subjects was performed. The leukotoxic activity on HL-60 cells was also evaluated. Leukotoxic activity was determined using a trypan blue exclusion method. The 530 bp deletion in the promoter region was evaluated by PCR using a PRO primer pair. A. actinomycetemcomitans was detected by culture and directly from crude subgingival biofilm by PCR using specific primers. By culture, A. actinomycetemcomitans was detected in nine (18 percent) of the periodontal patients and one (2 percent) healthy subject. However, by PCR, this organism was detected in 44 percent of the periodontal patients and in 16 percent of the healthy subjects. It was verified a great discrepancy between PCR detection of the ltx operon promoter directly from crude subgingival biofilm and from bacterial DNA. Only one periodontal sample harbored highly leukotoxic A. actinomycetemcomitans. Moreover, biotype II was the most prevalent and no correlation between biotypes and leukotoxic activity was observed. The diversity of leukotoxin expression by A. actinomycetemcomitans suggests a role of this toxin in the pathogenesis of periodontal disease and other infectious diseases

Aggregatibacter actinomycetemcomitans é um importante agente etiológico da periodontite e produz infecções extra-bucais. Neste estudo, foram detectados os biótipos, o gene ltxA associado à produção de leucotoxina e o promotor ltx em A. actinomycetemcomitans de pacientes com e sem periodontite. A atividade leucotóxica sobre células HL-60 também foi avaliada. A atividade leucotóxica foi determinada através do método de exclusão do azul de tripam. A deleção de 530 bp no promotor ltx foi avaliada usando-se o par de iniciadores PRO. A. actinomycetemcomitans foi detectado por cultura e por PCR. Por cultura, A. actinomycetemcomitans foi detectado em nove pacientes com periodontite (18 por cento) e em um indivíduo sadio (2 por cento). Por PCR esse microrganismo foi detectado em 44 por cento dos pacientes com periodontite e em 16 por cento dos saudáveis. Verificou-se diferença estatística entre a detecção do promotor do operon ltx, por PCR, diretamente do biofilme subgengival e do DNA bacteriano. Somente uma amostra clínica apresentou A. actinomycetemcomitans altamente leukotóxico. O biótipo II foi o mais prevalente e não foi observada correlação biótipo-atividade leucotóxica. A expressão da leucotoxina por A. actinomycetemcomitans na doença periodontal e outras doenças infecciosas necessita ser avaliado.

Occurrence of yeasts, enterococci and other enteric bacteria in subgingival biofilm of HIV-positive patients with chronic gingivitis and necrotizing periodontitis / Ocorrência de leveduras, enterococos e outras bactérias entéricas no biofilme subgengival de pacientes HIV-positivos com gengivite crônica e periodontite necrosante

The purpose of this study was to determine the prevalence of enteric bacteria and yeasts in biofilm of 80 HIV-positive patients with plaque-associated gingivitis or necrotizing periodontitis. Patients were subjected to extra, intra oral and radiographic examinations. The oral hygiene, bleeding on probing, gingival conditions, and attachment loss were evaluated. Clinical specimens were collected from gingival crevices or periodontal pockets, transferred to VMGA III, diluted and transferred to Sabouraud Dextrose agar with 100 µg/ml of chloramphenicol, peptone water, EVA broth, EMB agar, SS agar, Bile esculin agar and Brilliant green agar. Isolation of yeasts was carried out at room temperature, for 3-7 days; and for the isolation of enteric microorganisms plates were incubated at 37ºC, for 24-48 h. The yeasts identification was performed according to the carbon and nitrogen assimilation, fermentation of carbohydrates and germ tube formation. Bacteria were identified according to their colonial and cellular morphologies and biochemical tests. Yeasts were identified as Candida albicans and its occurrence was more common in patients with CD4+ below 200/mm³ and was affected by the extension of periodontal involvement (P = 0.0345). Enteric bacteria recovered from clinical specimens were identified as Enterobacter sakazakii, Enterobacter cloacae, Serratia liquefaciens, Klebsiella oxytoca and Enterococcus sp. Enterobacteriaceae and enterococci were detected in 32.5 percent of clinical samples from patients with necrotizing periodontitis. In conclusion, non-oral pathogenic bacteria and C. albicans were more prevalent in periodontal sites of HIV-positive patients with necrotizing periodontitis and chronic gingivitis.

O objetivo desse estudo foi avaliar a ocorrência de bactérias entéricas e leveduras no biofilme subgengival de pacientes HIV-positivos com gengivite crônica ou periodontite necrosante. Os pacientes foram submetidos a exame clínico e radiográfico e de higiene bucal, sangramento à sondagem, condições gengivais e a perda de inserção. Os espécimes clínicos de sulcos gengivais ou bolsas periodontais foram inoculados em ágar Sabouraud dextrose com 100 mg/ml de cloranfenicol, água peptonada, caldo EVA, ágar EMB, ágar SS, ágar Bile esculina e ágar verde brilhante. O cultivo de leveduras foi realizado à temperatura ambiente, de 3-7 dias; das enterobactérias a 37ºC de 24-48 h. A identificação das leveduras foi realizada pela assimilação de carbono e nitrogênio, fermentação de açucares e formação de tubo germinativo. As bactérias de acordo com a morfologia celular e colonial e testes bioquímicos. Foram identificadas Candida albicans e sua prevalência foi maior em pacientes com contagens de CD4+ < 200/mm³, e sua ocorrência foi afetada pela extensão da destruição periodontal (P = 0,0345). Enterobacteriaceae e enterococos foram detectados em 32,5 por cento dos pacientes com periodontite necrosante. As enterobactérias foram Enterobacter sakazakii, E. cloacae, Serratia liquefaciens, Klebsiella oxytoca e Enterococcus sp. Concluiu-se que bactérias patogênicas exógenas à cavidade bucal e C. albicans podem ser detectadas no biofilme subgengival de pacientes HIV-positivos com periodontite necrosante e gengivite.

Detection of putative periodontal pathogens in subgingival specimens of dogs

In this study, the presence of putative periodontal organisms, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, Fusobacterium nucleatum,Dialister pneumosintes,Actinobacillus actinomycetemcomitans,Campylobacter rectus,Eikenella corrodens and Treponema denticola were examined from subgingival samples of 40 dogs of different breeds with (25) and without (15) periodontitis, by using the PCR method. The PCR products of each species showed specific amplicons. Of the 25 dogs with periodontitis, P. gingivalis was detected in 16 (64 percent) samples, C. rectus in 9 (36 percent), A. actinomycetemcomitans in 6 (24 percent), P. intermedia in 5 (20 percent), T. forsythensis in 5 (20 percent), F. nucleatum in 4 (16 percent) and E. corrodens in 3 (12 percent). T. denticola and D. pneumosintes were not detected in clinical samples from dogs with periodontitis. Moreover, P. gingivalis was detected only in one (6.66 percent) crossbred dog without periodontitis. Our results show that these microorganisms are present in periodontal microbiota of dogs with periodontitits, and it is important to evaluate the role of these putative periodontal microorganisms play in the periodontitis in household pets particularly, dogs in ecologic and therapeutic terms, since these animals might acquire these periodontopahogens from their respective owners.

Neste estudo, a presença de patógenos periodontais, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, Fusobacterium nucleatum, Dialister pneumosintes, Actinobacillus actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens e Treponema denticola foi determinada por PCR, em amostras subgengivais de 40 cães com (25) e sem (15) doença periodontal. Os produtos amplificados pelo PCR para cada espécie bacteriana mostraram amplicons específicos. Dos 25 cães apresentando doença periodontal, P. gingivalis foi detectado em 16 amostras (64 por cento), C. rectus em 9 (36 por cento), A. actinomycetemcomitans em 6 (24 por cento), P. intermedia em 5 (20 por cento), T. forsythensis em 5 (20 por cento), F. nucleatum em 4 (16 por cento) e E. corrodens em 3 (12 por cento). Em nenhuma amostra clínica periodontal foi observada a presença de T. denticola ou D. pneumosintes. Adicionalmente, P. gingivalis foi detectado em apenas um (6,66 por cento) cão saudável, sem raça definida. Nossos resultados mostram que esses microrganismos estão presentes na microbiota periodontal de cães com periodontitis, e isto torna importante a avaliação do papel que esses microrganismos periodontais desempenham na periodontite de animais domésticos, particularmente cães, em termos ecológicos e terapêuticos, desde que esses cães podem adquirir esses periodontopatógenos de seus respectivos proprietários.

Ocorrência dos patógenos do complexo vermelho em sítios periodontais e periimplantares nas mesmas bocas / Occurrence of pathogens of the red complex in periodontal and peri-implant sites of the same mouths

O objetivo deste trabalho foi avaliar, pela reação em cadeia da polimerase (PCR), dirigida para as espécies do complexo vermelho (Porphyromonas gingivalis, Tannerella forsythensis e Treponema denticola), a ocorrência semiquantitativa desses patógenos em sulcos gengivaiscontrole (SG-C), bolsas periodontais (BP) e sulcos periimplantares (SPI) de cinco pacientesparcialmente desdentados portadores de implantes dentários havia mais de dois anos. Nos SG-C de três pacientes foi detectado o DNA de P. gingivalis, em um deles juntamente com o de T. forsythensis. Nas BP de todos, além de T. forsythensis e/ou T. denticola, foi constatada maior freqüência de P. gingivalis e a relação desses patógenos com a profundidade e o sangramentoà sondagem. Apesar da presença em sítios periodontais, nenhuma das espéciesalvo foi identificada nos SPI, embora quatro apresentassem sangramento à sondagem. Nossosresultados, obtidos em SPI sem periimplantite, confirmam que o método PCR possibilita um diagnóstico aplicável na análise de risco de doença, pois os patógenos da BP podem se translocar para os SPI, obrigando a um maior rigor no controle do biofilme dental.

Immunoglobulin G proteolytic activity of Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans produz protease ativa sobre imunoglobulina G humana, sendo um dos mecanismos importantes de escape do microrganismo. No presente trabalho, foi analisada a atividade proteolítica de sobrenadante de cultivo de A. actinomycetemcomitans ATCC 43718 sobre imunoglobulina G humana em função de concentração, tempo de cultivo do microrganismo e tempo de incubação com IgG, por ensaio imunoenzimático. Adicionalmente, foi determinada a fração com atividade de protease por meio de análise de eluatos de cromatografia em coluna de Sephadex G 150. Os resultados obtidos demonstraram que A. actinomycetemcomitans liberou protease ativa sobre imunoglobulina G humana em sobrenadante de cultivo, sendo a sua maior atividade evidenciada na concentração de 27,5 mg proteína/mL, com tempo de cultivo de 72 horas e com 24 horas de incubação com IgG. A massa molecular da fração ativa de protease foi compreendida entre 43 a 150 kDa.

Prevotella intermedia and Porphyromonas gingivalis isolated from osseointegrated dental implants: colonization and antimicrobial susceptibility

Neste estudo foram avaliadas a colonizacão e a susceptibilidade a antimicrobianos de P. intermedia e P. gingivalis isolados de amostras de sulcus gengivais e peri-implantares. As amostras foram coletadas de 30 pacientes submetidos a implantes, em três tempos diferentes: no momento da cirurgia, 20 e 60 dias após a instalacão do implante. Os organismos foram identificados por testes bioquímicos ou por kit comercial API 32-A e por PCR. A susceptibilidade antimicrobiana foi determinada usando-se o método de diluicão em ágar. Foram isolados dezenove P. intermedia (quatro de peri-implantites e 15 de sulco gengival) e somente sete P. gingivalis de sulco gengival. Pelo PCR os organismos foram detectados de sete amostras sete peri-implantares e de 32 gengivais. As bactérias foram susceptíveis aos antibióticos usados exceto para azitromicina com 65% de resistência para P. intermedia. As espécies avaliadas foram sensíveis para cádmio, níquel e paládio, e mostraram diferentes faixas de resistência para titânio, alumínio e bicloreto de mercúrio. A maioria de P. intermedia foi resistente para chumbo, prata, cobre, titânio, zinco, alumínio e bicloreto de mercúrio. As bactérias colonizaram implantes após 60 dias de cirurgia e PCR pode ser usado como ferramenta para a deteccão bacteriana na implantodontia.

Analysis of the presence of pathogens which predict the risk of disease at peri-implant sites through polymerase chain reaction (PCR)

A presença dos ADN de Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis e Prevotella intermedia em amostras coletadas de sulco periimplantar de 19 pacientes parcialmente desdentados foi analisada pela reação em cadeia da polimerase (PCR). Dentre esses 19 pacientes, dez apresentavam histórico de doença periodontal e nove não apresentavam antecedentes. Os resultados obtidos nesta análise foram relacionados com a profundidade do sulco periimplantar, o sangramento à sondagem e o provável risco de doença. Constatou-se que houve a amplificação do ADN das bactérias-alvo em sete amostras, sendo quatro de pacientes sem histórico de periodontopatia. Este resultado sugere que mesmo na ausência de sinais inflamatórios significantes, essa detecção qualitativa pode indicar risco de periimplantite, requerendo manutenção pós-operatória mais rigorosa.

Detection of pathogens from periodontal lesions

OBJETIVO: Realizar a detecção comparativa de A. actinomycetemcomitans e F. nucleatum de sítios periodontais e sadios. MÉTODOS: Foram analisadas amostras subgengivais de 50 pacientes com periodontite do adulto e de 50 indivíduos sadios. Ambos os organismos foram isolados em meio ágar de soja tripticaseína-bacitracina-vancomicina e detectados por PCR. Testes bioquímicos convencionais foram usados para a identificação bacteriana. RESULTADOS: A. actinomycetemcomitans e F. nucleatum foram isolados em 18 e 20por cento dos pacientes, respectivamente, e em 2 e 24por cento dos indivíduos sadios. Entre os isolados de A. actinomycetemcomitans, o biótipo II foi o mais prevalente. O par de iniciadores AA mostrou 100por cento de sensibilidade na detecção de A. actinomycetemcomitans, em ambos os grupos de indivíduos. Iniciadores ASH e FU foram também 100por cento sensíveis para detectar esse organismo em amostras de indivíduos sadios. O iniciador FN5047 foi mais sensível para detectar F. nucleatum em amostras de pacientes ou de sadios que o 5059S. Iniciadores ASH e 5059S foram mais específicos na detecção de A. actinomycetemcomitans e F. nucleatum, respectivamente, em amostras de pacientes e de sadios. CONCLUSÕES: PCR constitui-se uma ferramenta efetiva na detecção de patógenos periodontais de espécimes clínicos, fornecendo um diagnóstico rápido e seguro da doença periodontal. Entretanto, esse método depende da escolha dos iniciadores específicos utilizados.

Survey of antimicrobial susceptibility patterns of the bacteria of the Bacteroides fragilis group isolated from the intestinal tract of children

The bacteria of the Bacteroides fragilis group are considered important clinical pathogens and they are the most common anaerobes isolated from human endogenous infections. In this study, the susceptibility patterns to antibiotics and metals of 114 species of the B. fragilis group isolated from children with and without diarrhea were determined. Susceptibility was assayed by using an agar dilution method with Wilkins-Chalgren agar. All B. fragilis strains were resistant to lead and nickel, but susceptible to metronidazole and imipenem. beta-lactamase production was detected by using biological and nitrocefin methods, respectively, in 50 percent and 90.6 percent of the isolates of children with diarrhea and in 60 percent and 90 percent of the isolates of children without diarrhea. Our results show an increase of antibiotics and metals resistance in this microbial group, and a periodic evaluation of the antimicrobial susceptibility is needed. In Brazil, the contamination for antibiotics or metal ions is often observed, and it is suggested an increase the antimicrobial resistance surveillance of this microbial group, mainly those isolated from children's diarrhea.