Gram-negative Microbiota Research Articles

Gram-negative Microbiota Blooms in Premature Twins Discordant for Parenteral Nutrition-associated Cholestasis.

Parenteral nutrition-associated cholestasis (PNAC) causes serious morbidity in the neonatal intensive care unit. Infection with gut-associated bacteria is associated with cholestasis, but the role of intestinal microbiota in PNAC is poorly understood. We examined the composition of stool microbiota from premature twins discordant for PNAC as a strategy to reduce confounding from variables associated with both microbiota and cholestasis. Eighty-four serial stool samples were included from 4 twin sets discordant for PNAC. Random Forests was utilized to determine genera most discriminatory in classifying samples from infants with and without PNAC. In infants with PNAC, we detected a significant increase in the relative abundance of Klebsiella, Veillonella, Enterobacter, and Enterococcus (P < 0.05). Bray-Curtis dissimilarities in infants with PNAC were significantly different (P < 0.05) from infants without PNAC. Our findings warrant further exploration in larger cohorts and experimental models of PNAC to determine if a microbiota signature predicts PNAC, as a basis for future interventions to mitigate liver injury.

Antibiotic resistance and mobility of its genetic determinants in Lactobacillus fermentum

The work aimed to estimate the potential of transfer of resistance genes from Lactobacillus fermentum 5-1 to the Gram-negative intestinal microbiota. The sensitivity of antibiotics was assessed using the gradient agar method and microdilution method. Antibiotic resistance (AR) genes in bacterial genomic DNA were detected by PCR, followed by Sanger sequencing of the amplicons and BLASTn sequence analysis. Acinetobacter baumannii, Citrobacter freundii, and Escherichia coli had neither phenotypic nor genotypic resistance to tetracycline and therefore were selected among nine Gram-negative strains as recipients of the AR genes. The transferability of the AR genes from L. fermentum 5-1 to these bacteria was studied under cocultivation in a simulated colonic environment, in filter mating experiments, and using transformation of bacteria by electroporation. Based on the values of minimum inhibitory concentration (MIC), we found that this strain was resistant to vancomycin (MIC = 256 μg/mL), ciprofloxacin (MIC = 64 μg/mL), aminoglycosides (MIC = 54–256 μg/mL), and chloramphenicol (MIC = 8 μg/mL); sensitive to ampicillin (MIC = 0.5 μg/mL), rifampicin (MIC = 2 μg/mL), and cefotaxime (MIC = 0.12 μg/mL); and showed intermediate (low) resistance to erythromycin (MIC = 1 μg/mL) and tetracycline (MIC = 8 μg/mL). Using PCR analysis in the chromosomal DNA of L. fermentum 5-1, we detected the erythromycin resistance gene ermB, while two tetracycline resistance genes, tetM and tetK, were identified in plasmid DNA. Moreover, we demonstrated that L. fermentum 5-1 could transfer their tetK gene to C. freundii via electroporation and transformation with the plasmid DNA of the lactobacilli. Our results demonstrate that lactobacilli can negatively contribute to the problem of antibiotic resistance and warrant stricter control over the practical use of LAB.

THE USE OF INTRAVENOUS IMMUNOGLOBULIN PENTAGLOBIN IN THE COMPLEX TREATMENT OF SEVERE COMMUNITY-ACQUIRED PNEUMONIA IN CHILDREN

There are presented data from a comprehensive examination and treatment of 30 children with community-acquired pneumonia (CAP). For the treatment of young children with focal-confluent forms of CAP and the clinical manifestations of infectious-septic shock, Pentaglobin preparations containing IgM were used. Severe CAP with a tendency to destruction was more often observed in cases with the dominance of gram-negative flora in the etiological structure, most often detected in the foothill zone (18.2% of children), in the middle mountain zone - in- 13.3%, in the low-grade zone - in 5% of children. Pseudomonas aeruginosae was more commonly seeded in children in the foothill zone (12.5%), while in children living in the lowland and middle mountain zone in 2.5% and 6.6% correspondingly. The authors showed the preparation of intravenous immunoglobulin Pentoglobin is a safe, effective, easily tolerated means of etiotropic and pathogenetic therapy of severe CAP, caused more often by gram-negative and gram-positive bacterial microbiota.

Oxaliplatin-induced changes in microbiota, TLR4+ cells and enhanced HMGB1 expression in the murine colon.

Oxaliplatin is a platinum-based chemotherapeutic used for cancer treatment. Its use associates with peripheral neuropathies and chronic gastrointestinal side-effects. Oxaliplatin induces immunogenic cell death by provoking the presentation of damage associated molecular patterns. The damage associated molecular patterns high-mobility group box 1 (HMGB1) protein exerts pro-inflammatory cytokine-like activity and binds to toll-like receptors (namely TLR4). Gastrointestinal microbiota may influence chemotherapeutic efficacy and contribute to local and systemic inflammation. We studied effects of oxaliplatin treatment on 1) TLR4 and high-mobility group box 1 expression within the colon; 2) gastrointestinal microbiota composition; 3) inflammation within the colon; 4) changes in Peyer’s patches and mesenteric lymph nodes immune populations in mice. TLR4+ cells displayed pseudopodia-like extensions characteristic of antigen sampling co-localised with high-mobility group box 1 -overexpressing cells in the colonic lamina propria from oxaliplatin-treated animals. Oxaliplatin treatment caused significant reduction in Parabacteroides and Prevotella1, but increase in Prevotella2 and Odoribacter bacteria at the genus level. Downregulation of pro-inflammatory cytokines and chemokines in colon samples, a reduction in macrophages and dendritic cells in mesenteric lymph nodes were found after oxaliplatin treatment. In conclusion, oxaliplatin treatment caused morphological changes in TLR4+ cells, increase in gram-negative microbiota and enhanced HMGB1 expression associated with immunosuppression in the colon.

Listeria monocytogenes strains show large variations in competitive growth in mixed culture biofilms and suspensions with bacteria from food processing environments

Interactions and competition between resident bacteria in food processing environments could affect their ability to survive, grow and persist in microhabitats and niches in the food industry. In this study, the competitive ability of L. monocytogenes strains grown together in separate culture mixes with other L. monocytogenes (L. mono mix), L. innocua (Listeria mix), Gram-negative bacteria (Gram− mix) and with a multigenera mix (Listeria + Gram− mix) was investigated in biofilms on stainless steel and in suspensions at 12 °C. The mixed cultures included resident bacteria from processing surfaces in meat and salmon industry represented by L. monocytogenes (n = 6), L. innocua (n = 5) and Gram-negative bacteria (n = 6; Acinetobacter sp., Pseudomonas fragi, Pseudomonas fluorescens, Serratia liquefaciens, Stenotrophomonas maltophilia). Despite hampered in growth in mixed cultures, L. monocytogenes established in biofilms with counts at day nine between 7.3 and 9.0 log per coupon with the lowest counts in the Listeria + G− mix that was dominated by Pseudomonas. Specific L. innocua inhibited growth of L. monocytogenes strains differently; inhibition that was further enhanced by the background Gram-negative microbiota. In these multispecies and multibacteria cultures, the growth competitive effects lead to the dominance of a strong competitor L. monocytogenes strain that was only slightly inhibited by L. innocua and showed strong competitive abilities in mixed cultures with resident Gram-negative bacteria. The results indicates complex patterns of bacterial interactions and L. monocytogenes inhibition in the multibacteria cultures that only partially depend on cell contact and likely involve various antagonistic and bacterial tolerance mechanisms. The study indicates large variations among L. monocytogenes in their competitiveness under multibacterial culture conditions that should be considered in further studies towards understanding of L. monocytogenes persistence in food processing facilities.

Porphyromonas gingivalis-Derived Lipopolysaccharide Combines Hypoxia to Induce Caspase-1 Activation in Periodontitis.

Periodontitis is defined as inflammation affecting the supporting tissue of teeth. Periodontal pathogens initiate the disease and induce inflammatory host response. Hypoxia may accelerate the process by producing pro-inflammatory factors. The aim of this study is to investigate the effect of Porphyromonas gingivalis (P. gingivalis) lipopolysaccharides (LPS) and Escherichia coli (E. coli) LPS in inducing caspase-1 activation in normoxic or hypoxic phases. The results showed that healthy gingiva was in a normoxic phase (HIF-1α negative). However, hypoxia appeared in periodontitis, in which NLRP3, cleaved-caspase-1, interleukin 1 beta (IL-1β) and caspase-1-induced cell death was enhanced in periodontitis specimens. The in vitro experiment showed that P. gingivalis LPS slightly decreased the level of NLRP3 and IL-1β in gingival fibroblasts under normoxia. Surprisingly, hypoxia reversed the effects of P. gingivalis LPS, highly promoted caspase-1 activation and IL-1β maturation. E. coli LPS, a kind of pathogen-associated molecular pattern (PAMP) was chosen to simulate the effect of Gram-negative microbiota. Different from P. gingivalis LPS, E. coli LPS enhanced IL-1β maturation both in normoxia and hypoxia. Moreover, E. coli LPS turned normoxia into hypoxia phase in experimental periodontitis model, which may subsequently propel the inflammatory effect of P. gingivalis LPS. It was concluded that E. coli LPS induced a hypoxic phase, which is a combing pathological factor of P. gingivalis LPS in caspase-1 activating and IL-1β maturation in periodontal inflammation.

Thermoneutral housing exacerbates nonalcoholic fatty liver disease in mice and allows for sex-independent disease modeling.

Non-alcoholic fatty liver disease (NAFLD), a common prelude to cirrhosis and hepatocellular carcinoma, is the most common chronic liver disease worldwide. Defining the molecular mechanisms underlying the pathogenesis of NAFLD has been hampered by a lack of animal models that closely recapitulate the severe end of the human disease spectrum, including bridging hepatic fibrosis. Here, we demonstrate that a novel experimental model employing thermoneutral housing, as opposed to standard housing, resulted in lower stress-driven production of corticosterone, augmented mouse proinflammatory immune responses and markedly exacerbated high fat diet (HFD)-induced NAFLD pathogenesis. Disease exacerbation at thermoneutrality was conserved across multiple mouse strains and was associated with augmented intestinal permeability, an altered microbiome and activation of inflammatory pathways associated with human disease. Depletion of Gram-negative microbiota, hematopoietic cell deletion of Toll-like receptor 4 (TLR4) and inactivation of the interleukin-17 (IL-17) axis resulted in altered immune responsiveness and protection from thermoneutral housing-driven NAFLD amplification. Finally, female mice, typically resistant to HFD-induced obesity and NAFLD, develop full-blown disease at thermoneutrality. Thus, thermoneutral housing provides a sex-independent model of exacerbated NAFLD in mice and represents a novel approach for interrogation of the cellular and molecular mechanisms underlying disease pathogenesis.

A method for culturing Gram-negative skin microbiota.

BackgroundCommensal Gram-negative (CGN) microbiota have been identified on human skin by DNA sequencing; however, methods to reliably culture viable Gram-negative skin organisms have not been previously described.ResultsThrough the use of selective antibiotics and minimal media we developed methods to culture CGN from skin swabs. We identified several previously uncharacterized CGN at the species level by optimizing growth conditions and limiting the inhibitory effects of nutrient shock, temperature, and bacterial competition, factors that may have previously limited CGN isolation from skin cultures.ConclusionsOur protocol will permit future functional studies on the influences of CGN on skin homeostasis and disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0684-9) contains supplementary material, which is available to authorized users.

Proteobacteria from the human skin microbiota: Species-level diversity and hypotheses

The human skin microbiota is quantitatively dominated by Gram-positive bacteria, detected by both culture and metagenomics. However, metagenomics revealed a huge variety of Gram-negative taxa generally considered from environmental origin. For species affiliation of bacteria in skin microbiota, clones of 16S rRNA gene and colonies growing on diverse culture media were analyzed. Species-level identification was achieved for 81% of both clones and colonies. Fifty species distributed in 26 genera were identified by culture, mostly belonging to Actinobacteria and Firmicutes, while 45 species-level operational taxonomic units distributed in 30 genera were detected by sequencing, with a high diversity of Proteobacteria. This mixed approach allowed the detection of 100% of the genera forming the known core skin Gram-negative microbiota and 43% of the known diversity of Gram-negative genera in human skin. The orphan genera represented 50% of the current skin pan-microbiota. Improved culture conditions allowed the isolation of Roseomonas mucosa, Aurantimonas altamirensis and Agrobacterium tumefaciens strains from healthy skin. For proteobacterial species previously described in the environment, we proposed the existence of skin-specific ecotypes, which might play a role in the fine-tuning of skin homeostasis and opportunistic infections but also act as a shuttle between environmental and human microbial communities. Therefore, skin-associated proteobacteria deserve to be considered in the One-Health concept connecting human health to the health of animals and the environment.

Abstract P4-04-07: Breast cancer tissue context determines whether inflammation with bacterial/psoriasin signature is pro- or anti-carcinogenesis

Abstract Objective: It is often neglected that breast tissue microbiota and systematic bacterial infection actively and potently influence the development of breast cancer. With reports of both pro-cancer and anti-cancer roles of bacterial inflammation, the actual effect of bacterial inflammation to breast cancer and its mechanism remain unclear and are of our interests. Rationale: Different from other human tissues, breast tissue microbiota is majorly Gram-negative at both normal and pathological statuses. We discovered that bacterial inflammation in breast tissue has a signature of Psoriasin (S100A7) impact which resembles psoriasis of the skin. However, this Psoriasin mediated inflammation can either promote or inhibit breast cancer development. We hypothesize that different tissue context determines the differential effects of bacterial/Psoriasin inflammation on breast cancer development. Results: We first verified the presence of commensal Gram-negative microbiota (and LPS) in breast tissues under normal and pathological circumstances using mouse models. We observed that in both normal and cancerous breast tissues, bacterial factors (such as LPS) found in breast triggered secretion of Psoriasin by mammary adenocytes and Psoriasin mediated inflammation. This type of inflammation is featured by macrophage recruitment and ductal infiltration induced by Psoriasin. However, in normal tissues, macrophages matured into M1 type (iNOS positive); whereas in cancerous tissues, macrophages matured into M2 type (Arginase-1 positive). In normal tissues, Psoriasin and M1 caused an unresolved accumulation of inflammation in mammary ducts without lactation, which might lead to accelerated cancer initiation. In tumors, Psoriasin and M2 increased tumor growth and metastasis in immune-competent mammary epithelial specific expression mouse models. This was further confirmed in human patient samples and large cohort bioinformatic analysis. Using human breast cancer cell lines and nude mice, we elucidated that inflammation related Psoriasin upregulation and secretion in cancer cells had differential effects on tumor growth depending on its tissue source. Psoriasin increased cell proliferation and tumor growth in basal-like cancer cells; whereas it decreased cell proliferation and tumor growth in non-basal-like cancer cells. And this difference was mediated through differential regulation of the NF-κB/miR-29b/p53 pathway. in vitro molecular study and in vivo models verified the important role of miR-29b switch in the differential effects of Psoriasin in different tissue types of breast cancer cells. Conclusion: Bacterial/Psoriasin inflammation differentially influences breast cancer development depending on the tissue context, and it involves the effects on macrophages and cancer cells. Our work provides novel potentials to improving prognosis and targeting inflammation to treat breast cancer in personalized medicine. Citation Format: Helong Zhao, Mohd W Nasser, Tasha Wilkie, Ramesh K Ganju. Breast cancer tissue context determines whether inflammation with bacterial/psoriasin signature is pro- or anti-carcinogenesis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-07.

Pulp and plaque microbiotas of children with severe early childhood caries

Background and objective: Bacterial invasion into pulps of primary teeth can lead to infection and premature tooth loss in children. This pilot study aimed to explore whether the microbiota of carious exposures of dental pulps resembles that of carious dentin or that of infected root canals.Design: Children with severe early childhood caries were studied. Children were consented and extent of caries, plaque, and gingivitis measured. Bacteria were sampled from carious lesion biofilms and vital carious exposures of pulps, and processed by anaerobic culture. Isolates were characterized from partial sequences of the 16S rRNA gene and identified by comparison with taxa in the Human Oral Microbiome Database (http://www.HOMD.org). The microbiotas of carious lesions and dental pulps were compared using univariate and multivariate approaches.Results: The microbiota of cariously exposed pulps was similar in composition to that of carious lesion biofilms except that fewer species/taxa were identified from pulps. The major taxa identified belonged to the phyla Firmicutes (mainly streptococci) and Actinobacteria (mainly Actinomyces species). Actinomyces and Selenomonas species were associated with carious lesions whereas Veillonella species, particularly Veillonella dispar was associated with pulps. Other bacteria detected in pulps included Streptococcus mutans, Parascardovia denticolens, Bifidobacterium longum, and several Lactobacillus and Actinomyces species. By principal, component analysis pulp microbiotas grouped together, whereas those in caries biofilms were widely dispersed.Conclusions: We conclude that the microbiota of cariously exposed vital primary pulps is composed of a subset of species associated with carious lesions. Vital primary pulps had a dominant Firmicutes and Actinobacteria microbiota which contrasts with reports of endodontic infections which can harbor a gram-negative microbiota. The microbiota of exposed primary pulps may provide insight into bacterial species at the forefront of caries invasion in dentinal lesions that can invade into the pulp and the nature of species that need suppressing for successful pulp therapy.

Bioprospecting of culturablebactéria with potential polyhydroxyalkanoates (PHA) producer, isolated from contaminated sites of Manaus-AM/Brazil

Polyethylene and polypropylene, known as conventional plastic, from non-renewable sources, like oil, are successfully replacing by glasses and papers in packaging industry due to their low cost, variety and durability [1]. However, these plastics for being slow degradability and hinder the exchange of gas and the decomposition of other compounds cause health problems when they are not disposed of properly [2]. Nowadays, there are another renewable plastic sources, known as polyhydroxyalkanoates (PHA), manufactured by plants, fungi and/or bacteria source. From the ecological view this plastics are important because their degradation are faster and requires less energy in their manufacture processes, reducing significantly the CO2 emissions [3]. The subject of this work was a bioprospect culturable bacteria potentially producers of PHAs, from water and plant species contaminated from anthropogenic actions. Bacteria were isolated from two plants (Superfamily Legominosae) and water collected from two points of Forty affluent of the city of Manaus - Amazonas. For the isolation of the water samples, 50 l were grown in two culture media (Nutrient Agar Agar and Lb) containing 50 mg / mL of fluconazole at 26 oC for 7 days. The plant samples (leaf, stem and root) were subjected to aseptic procedure described by Souza et al, 2004 [4]. Isolated bacteria were purified by the method of cross-striations and then evaluated by analyzing the morphology of the Gram stain. For detection of potentially producing PHA bacteria it was used the test of Sudan Black plating cell culture with 24 wells containing YM medium and incubated at 26°C for 72 hours according to the protocol described by the National Center for Biotechnology Education (2012 [5]). Of the four samples collected at two points of Igarape do Quarenta, 80 bacteria were isolated, of which 64 were randomly selected and evaluated for potential production of PHA. From all bacteria evaluated, 13 were positive (30%), six (9.37%) of the plant #2, three (4.71%) of the plant #1 and two (3.12%) of each point of colleted water. From the 13 positive results, 12 were characterized as Gram negative and Gram negative as streptococci isolated from the root of the plant 1. This research showed a predominantly Gram-negative microbiota, whose endophytic bacteria were the main source, compared with water of potentially producing of PHA bacteria and therefore the bacteria present in polluted waters and adjacent plants of Igarape do Quarenta in the city of Manaus may be considered potentially producing PHA.

Changes in fecal microbiota of healthy dogs administered amoxicillin

The effect of oral amoxicillin treatment on fecal microbiota of seven healthy adult dogs was determined with a focus on the prevalence of bacterial antibiotic resistance and changes in predominant bacterial populations. After 4-7 days of exposure to amoxicillin, fecal Escherichia coli expressed resistance to multiple antibiotics when compared with the pre-exposure situation. Two weeks postexposure, the susceptibility pattern had returned to pre-exposure levels in most dogs. A shift in bacterial populations was confirmed by molecular fingerprinting of fecal bacterial populations using denaturing gradient gel electrophoresis (PCR-DGGE) of the 16S V3 rRNA gene region. Much of the variation in DGGE profiles could be attributed to dog-specific factors. However, permutation tests indicated that amoxicillin exposure significantly affected the DGGE profiles after controlling for the dog effect (P=0.02), and pre-exposure samples were clearly separated from postexposure samples. Sequence analysis of DGGE bands and real-time PCR quantification indicated that amoxicillin exposure caused a shift in the intestinal ecological balance toward a Gram-negative microbiota including resistant species in the family Enterobacteriaceae.

3-Hydroxy fatty acids in faeces of patients with Crohn's disease or ulcerative colitis as a non-invasive diagnostic approach

The microbiota is part of the gastrointestinal ecosystem. A more detailed understanding should provide insight into multiple human disease states. This study investigated inflammatory bowel disease (Crohn’s disease and ulcerative colitis). Previous analyses have suggested a role of gram-negative bacteria including Escherichia coli . An integrated procedure is presented where gas chromatography-mass spectrometry is used to determine chemical markers of gram-negative bacterial lipopolysaccharide (3-hydroxy fatty acids with 1018 carbon atoms) in faecal samples. Six patients with Crohn’s disease (CD), five with ulcerative colitis (UC) and six healthy adults were chosen as groups of interest. Nine saturated straight-chain 3-OH fatty acids of 10-18 carbon chain lengths and six iso- and anteiso-branched-chain 3-OH fatty acids of 15-18 carbon chain lengths were detected. Significant differences were found in the 3-OH n-C 17:0 , 3-OH i-C 18:0 and 3-OH n-C 18:0 composition of faeces. The present study therefore confirms that alteration of the composition of the endogenous gramnegative microbiota may be of importance in inflammatory bowel disease and those alterations could be detected with a non-invasive chemical-analytical approach. Key words: faecal microbiota, gas chromatography-mass spectrometry, inflammatory bowel diseases

Qualidade microbiológica de leite cru refrigerado e isolamento de bactérias psicrotróficas proteolíticas

A estocagem do leite cru refrigerado na fonte de produção reduz perdas econômicas por atividade acidificante de bactérias mesofílicas, mas permite a seleção de bactérias psicrotróficas relacionadas a problemas tecnológicos e econômicos na indústria de laticínios. Com o objetivo de avaliar a qualidade microbiológica de leite cru, foram analisadas amostras provenientes de tanques de refrigeração individual, coletivos e do silo de uma indústria processadora de leite. Além disso, bactérias psicrotróficas proteolíticas foram isoladas do leite cru refrigerado e caracterizadas quanto à reação ao Gram e fermentação de glicose. O leite cru refrigerado mantido no silo industrial não atendeu ao padrão microbiológico legal e apresentou contagens microbianas significativamente superiores às do leite mantido em tanques individuais ou coletivos. Diferença significativa na contaminação por mesófilos e psicrotrófilos proteolíticos e não proteolíticos e por Pseudomonas foi observada entre as amostras coletadas nos tanques de refrigeração e no silo industrial. A microbiota Gram-negativa foi isolada com maior freqüência, especialmente bactérias Gram-negativas não fermentadoras de glicose.

Bone powder enhances the effectiveness of bioactive glass S53P4 against strains of Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans in suspension

Objective. To assess whether bone powder in suspension enhances the antimicrobial efficacy of bioactive glass S53P4 against Gram-negative microbiota commonly associated with peri-implant disease. Methods. Standardized suspensions of Porphyromonas gingivalis ATCC 33277 and YH 3, as well as Actinobacillus actinomycetemcomitans ATCC 29523 and KK 2 were added to 24-h suspensions of bioactive glass S53P4 with ground bovine bone powder, decalcified bone, or hydroxylapatite powder. Recovery of viable bacteria was assessed using anaerobic culture methods. As a reference, the antibacterial effect of an inert borosilicate powder with a particle size corresponding to that of the bioactive glass was tested. Counts of bacteria suspended in a pure unbuffered saline solution served as controls. Results. A significant drop in viable microorganisms was observed in suspensions of bioactive glass and bone powder compared to counterparts of pure bioactive glass. In contrast, neither the presence of hydroxylapatite powder nor the presence of decalcified bone in suspension caused any increase in bioactive glass killing efficacy on the microorganisms under investigation. Inert borosilicate glass showed no antibacterial effects per se or in combination with bone powder. Conclusion. The antimicrobial effect of a combined bioactive glass-ground bone powder suspension was an in vitro observation which should be confirmed using adequate in vivo models.

Microbiota of health, gingivitis, and initial periodontitis

This study compared the subgingival microbiota in periodontal health, gingivitis and initial periodontitis using predominant culture and a DNA probe, checkerboard hybridization method. 56 healthy adult subjects with minimal periodontal attachment loss were clinically monitored at 3-month intervals for 12 months. More sites demonstrated small increments of attachment loss than attachment gain over the monitoring period. Sites, from 17 subjects, showing > or = 1.5 mm periodontal attachment loss during monitoring were sampled as active lesions for microbial analysis. Twelve subjects demonstrated interproximal lesions, and 5 subjects had attachment loss at buccal sites (recession). Cultural studies identified Bacteroides forsythus, Campylobacter rectus, and Selenomonas noxia as the predominant species associated with active interproximal lesions (9 subjects), whereas Actinomyces naeslundii, and Streptococcus oralis, were the dominant species colonizing buccal active sites. A. naeslundii, Campylobacter gracilis, and B. forsythus (at lower levels than active sites) were the dominant species cultured from gingivitis (10 subjects). Health-associated species (10 subjects) included Streptococcus oralis, A. naeslundii, and Actinomyces gerencseriae. DNA probe data identified higher mean levels of B. forsythus and C. rectus with active (7 subjects) compared to inactive periodontitis sites. Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans were detected infrequently. Cluster analysis of the cultural microbiota grouped 8/9 active interproximal lesions in one subcluster characterized by a mostly gram-negative microbiota, including B. forsythus and C. rectus. The data suggest that B. forsythus C. rectus and S. noxia were major species characterizing sites converting from periodontal health to disease. The differences in location and microbiota of interproximal and buccal active sites suggested that different mechanisms may be involved in increased attachment loss.

The Role ofEubacteriumSpecies in Periodontal Disease and Other Oral Infections

Periodontal disease has been traditionally associated with an anaerobic gram-negative microbiota. However, improvements in sampling handling and culture media have revealed that gram-positive anaerobes belonging to the genus Eubacterium can make up around half of the microbiota in advanced disease. The asaccharolytic species E. brachy, E. nodatum, E. saphenum and E. timidum show significant associations with severe periodontal disease, but are only rarely found in oral health. Antibodies to E. brachy and E. timidum are raised in patients with periodontal disease compared with healthy controls. Studies of the genus are hampered by the poor and slow growth of the majority of species and the indifferent taxonomy of the group. It is estimated that there are at least 20 un-named taxa. 16S ribosomal RNA sequencing has revealed that the oral asaccharolytic Eubacterium species are closely related to the genera Clostridium and Peptostreptococcus and exhibit three distinct lines of descent. Virulence factors produced by these organisms are as yet unknown although one unnamed taxon which is strongly associated with dento-alveolar abscesses has been established in continuous culture and has been shown to exhibit a wide range of aminopeptidase activity. Keywords: Eubacterium , periodontitis.

Virulence Properties of Escherichia coli Isolated from the Rectal Mucosa of Patients with First Attacks of Colitis

The aerobic gram-negative microbiota and virulence properties of Escherickia coli were studied in 157 rectal biopsy specimens from 50 patients with a first attack of inflammatory bowel disease (IBD) and in 56 such specimens from 20 patients with infectious colitis or infectious-type colitis (non-relapsing colitis, NRC). E. coli strains that produced both haemolysin, enterohaemolysin or colicin were found to a similar extent in IBD and NRC. E. coli strains carrying recognised virulence factors, such as enterotoxins and adhesins, were rarely isolated from patients of either group. No property was found more often in active than in inactive phases of the disease. In addition, the properties of E. coli were evenly distributed between ulcerative colitis and Crohn’s disease. E. coli strains belonging to classical enteropathogenic or enteroinvasive serotypes were not isolated. Non-motile E. coli lacking H antigen were isolated to a similar extent in IBD and NRC. Repeated isolation of the same serotype of E. coli was more common in IBD (P<0.05). Keywords: Colitis; Inflammatory bowel disease; Ulcerative colitis; Crohn’s disease; Infectious colitis; Infectioustype colitis; E. coli ; Haemolysin; Invasiveness; Serotype; Virulence; Enterotoxin.

Factors in Virulence Expression and Their Role in Periodontal Disease Pathogenesis

The classic progression of the development of periodontitis with its associated formation of an inflammatory lesion is characterized by a highly reproducible microbiological progression of a Gram-positive microbiota to a highly pathogenic Gram-negative one. While this Gram-negative microbiota is estimated to consist of at least 300 different microbial species, it appears to consist of a very limited number of microbial species that are involved in the destruction of periodontal diseases. Among these "putative periodontopathic species" are members of the genera Porphyromonas, Bacteroides, Fusobacterium, Wolinella, Actinobacillus, Capnocytophaga, and Eikenella. While members of the genera Actinomyces and Streptococcus may not be directly involved in the microbial progression, these species do appear to be essential to the construction of the network of microbial species that comprise both the subgingival plaque matrix. The temporal fluctuation (emergence/disappearance) of members of this microbiota from the developing lesion appears to depend upon the physical interaction of the periodontal pocket inhabitants, as well as the utilization of the metabolic end-products of the respective species intimately involved in the disease progression. A concerted action of the end-products of prokaryotic metabolism and the destruction of host tissues through the action of a large number of excreted proteolytic enzymes from several of these periodontopathogens contribute directly to the periodontal disease process.(ABSTRACT TRUNCATED AT 250 WORDS)