Effect of Actinobacillus actinomycetemcomitans, Wolinella recta and Bacteroides gingivalis on the viability of retinoic acid-induced and dimethyl sulfoxide-induced HL-60 cells

We studied the interactions between viable and heat-killed, opsonized and unopsonized periodontopathic bacteria with both uninduced and induced HL-60 promyelocytic leukemia cells. The cells were induced to differentiate into granulocyte-like cells by incubation with retinoic acid (RA) or dimethyl sulfoxide (DMSO). When unopsonized, Wolinella recta ATCC 33228 significantly suppressed the net proliferation of uninduced HL-60 cells, Actinobacillus actinomycetemcomitans strain Y4 was markedly lethal to the cells, and Bacteroides gingivalis ATCC 33277 had no effect. Unopsonized and opsonized A. actinomycetemcomitans and W. recta had equally potent lethal effects on induced HL-60 cells. Unopsonized B. gingivalis was not lethal to the induced cells in the dose used (100 bacteria/HL-60 cell), but opsonized B. gingivalis was lethal, especially in the first 24 h. The killing effects of A. actinomycetemcomitans and W. recta were largely eliminated if they were heated (56 degrees C, 30 min) before being added to the induced HL-60 cells. RA-induced HL-60 cells were more sensitive to the lethal effects of A. actinomycetemcomitans and W. recta than were DMSO-induced cells. The results suggest that the HL-60 cell line may be a useful model for studying granulocyte-bacteria interactions.     

Fatty acids and sugars in lipopolysaccharides from Bacteroides intermedius, Bacteroides gingivalis and Bacteroides loescheii

Lipopolysaccharide (LPS, endotoxin) was isolated from Bacteroides intermedius, Bacteroides gingivalis and Bacteroides loescheii by phenol extraction, purified by ultracentrifugation and investigated chemically by gas chromatography and mass spectrometry. Twenty different fatty acids were found. The fatty acid composition differed between the bacterial species, and intraspecies variation was also recorded. All endotoxins contained 3-hydroxy-15-methylhexadecanoic acid and 3-hydroxy-hexadecanoic acid as major hydroxy fatty acids. Additional fatty acids were straight chain or iso- or anteiso-branched fatty acids, and straight chain or iso-branced hydroxylated fatty acids. The sugar composition showed great variability between different strains of the same species. Heptose and 2-keto-3-deoxyoctonate (KDO) were detected in LPS from strains of B. gingivalis and B. intermedius. A tentatively identified glucosamine disaccharide was detected in all three Bacteroides species. This is indicative of lack of phosphorylation in the lipid A backbone, which is in concordance with the low general toxicity of Bacteroides LPS.     

Isolation and characterization of plasmid DMA from Bacteroides strains isolated from the human oral cavity

A total of 134 Bacteroides strains isolated from the human oral cavity were examined for plasmid content. Plasmid DNA was isolated from 7 strains of black-pigmented Bacteroides , including Bacteroides levii strains ATCC 29147 and JP2. All isolated Bacteroides plasmid DNA had a molecular mass of 5.9 M daltons and showed the same electrophoretic pattern when treated with restriction endonuclease. No positive correlation was found between plasmid DNA content and antibiotic resistance, bacteroiocinogenic activity or hemagglutinating activity.     

Hydrophobicities of human polymorphonuclear leukocytes and oral Bacteroides and Porphyromonas spp., Wolinella recta, and Eubacterium yurii with special reference to bacterial surface structures

Abstract – The hydrophobicities of human polymorphonuclear leukocytes (PMNLs) and Bacteroides buccae, B. oris, B. oralis, B.veroralis, B. buccalis, B. heparinolyticus, B. intermedius, B. denticola, B. loescheii, B. melaninogenicus, Porphyromonas gingivalis, P. endodontalis, Wolinella recta , and Eubacterium yurii were studied by the hexadecane method. The majority of the strains were equally or less hydrophobic than the PMNLs. Only in the case of E. yurii and the only strain of B. buccalis were all strains more hydrophobic than the PMNLs. However, some strains of B. intermedius , B. oris , B. denticola , and P. gingivalis were also more hydrophobic than the PMNLs. With the exception of B. intermedius and species with a crystalline surface protein layer (S-layer), the strains of all other species with a thick capsule were more hydrophilic than the strains with little or no extracellular polymeric material. All strains of the S-layer species were either quite hydrophilic or hydrophobic depending on the species, totally irrespective of the presence of the capsule. The results suggest that the S-layers of oral anaerobic bacteria may be important determinants of cell surface hydrophobicity.     

Direct and immune-cell-mediated effects of Bacteroides gingivalis on bone metabolism in vitro

We investigated direct and immune-cell-mediated effects of Bacteroides gingivalis on bone metabolism in vitro. Fetal mouse long-bone rudiments were cultured under aerobic conditions in the presence of (a) intact bacteria, (b) low molecular weight (MW less than 1000) metabolic products of the bacteria, or (c) conditioned media of mouse spleen cells activated by whole bacteria. A suspension of intact bacteria, added directly to the bone culture, had no effect on bone resorption or bone formation. Low molecular weight (MW less than 1000) excretion products of the bacteria inhibited bone resorption and transiently reduced mineralization of the diaphysis, while the growth in length of the bones was not affected. However, conditioned media of bacteria-activated spleen cells strongly enhanced bone resorption and increased osteoclast numbers in the bone culture, while inhibiting mineral formation in the diaphysis. This led to a strongly negative mineral balance. These data do not support a direct effect of either bacteria or bacterial products on bone tissue as a likely explanation for bone loss in periodontal disease. Rather, they favour the concept that the loss of bone in this disease is an indirect effect of the host response, resulting from the contact of immune cells with the bacteria. This implies that bacterial invasion of the connective tissue of the gingiva may not be a prerequisite for alveolar bone loss.     

PCR ribotyping and arbitrarily primed PCR for the comparison of enterotoxigenic Bacteroides fragilis strains from two Polish university hospitals

Objective: To study the clinical incidence and possible clonal relatedness of enterotoxigenic strains of Bacteroides fragilis among pediatric and adult patients in two Polish university hospitals.
Method: Fecal samples from 201 adults and 131 infants (with or without diarrhea) and vaginal samples from 100 pregnant women nursed in two Polish university hospitals were analyzed with respect to carriage of enterotoxin-producing Bacteroides fragilis (ETBF). This putative pathogen was identified by cultivation and subsequent cytopathogenicity testing of culture supernatants on HT/29 C1 cells.
Results and discussion: Two ETBF strains were isolated from childrens' feces; two additional strains were isolated from adults, and from the vaginal samples only a single strain was isolated. One strain (W2) was isolated from a child with diarrhea. These incidence figures, the fact that all ETBF isolates were shown to produce strongly differing amounts of the cytotoxin, and the genetic unrelatedness of the strains as demonstrated by two different PCR-mediated DNA typing procedures, indicates that clonal spread of ETBF is presently not a clinical problem in these hospitals. It was shown that PCR-mediated ribotyping and arbitrarily primed PCR can be applied with success to study the epidemiology of ETBF.     

Population structure and distribution of virulence-related genes of Bacteroides fragilis isolates from Korea and Japan

Sequences for rpoB, gyrB, pdiA, and ompA were determined from 63 Bacteroides fragilis isolates, which were from Korea and Japan and include 4 reference strains. All 4 gene sequences supported clear separation of the cfi⁺ group from the cfi⁻ group. Combined sequences of the 60 division I isolates (cfi⁻) produced 45 different clones. Apparent discordance of gene trees, index of association, maximum likelihood test, and homoplasy ratio all supported a high frequency of recombination. There was no association between the presence of virulence-related genes and phylogenetic clustering in any gene tree.     

Molecular detection and identification of anaerobic bacteria

Anaerobic bacteria are organisms which are inactivated by exposure to air, and some species grow very slowly. Thus, they are good candidates for direct detection and culture confirmation using molecular diagnostic techniques. In clostridial species, polymerase chain reaction (PCR) amplification techniques have been used to demonstrate a segment of the gene encoding neurotoxins, enterotoxins, or cytotoxins. Using these techniques,Clostridium botulinum strains were directly isolated and toxin-typed from food samples.Clostridium tetani strains were identified, and toxigenicClostridium difficile strains and enterotoxin-producingClostridium perfringens strains were either confirmed in culture studies or directly detected from stool or food specimens. Among nonclostridial anaerobic bacteria,Propionibacterium acnes, Mobiluncus, gram-negative periodontal pathogens includingPorphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Bacteroides forsythus, Bacterioides fragilis and enterotoxin-producingB. fragilis, Fusobacterium prausnitzii (a common constituent of fecal flora), andBilophila wadsworthia have been studied on a molecular level using oligonucleotide probes and PCR methods. Universal PCR primers may be the choice for differentiation of anaerobes which are commonly found in polymicrobial infections and quantitative nucleic acid amplification technology has been used extensively in these situations. However, some studies lack a sufficient number of strains tested to evaluate the reliability of these molecular methods. The accumulation of additional data using these techniques is crucial to their widespread use or to estimate their limitations.     

Linking fecal bacteria in rivers to landscape,geochemical, and hydrologic factors and sources at the basin scale

Linking fecal indicator bacteria concentrations in large mixed-use watersheds back to diffuse human sources, such as septic systems, has met limited success. In this study, 64 rivers that drain 84% of Michigan’s Lower Peninsula were sampled under baseflow conditions for Escherichia coli, Bacteroides thetaiotaomicron (a human source-tracking marker), landscape characteristics, and geochemical and hydrologic variables. E. coli and B. thetaiotaomicron were routinely detected in sampled rivers and an E. coli reference level was defined (1.4 log₁₀ most probable number⋅100 mL⁻¹). Using classification and regression tree analysis and demographic estimates of wastewater treatments per watershed, septic systems seem to be the primary driver of fecal bacteria levels. In particular, watersheds with more than 1,621 septic systems exhibited significantly higher concentrations of B. thetaiotaomicron. This information is vital for evaluating water quality and health implications, determining the impacts of septic systems on watersheds, and improving management decisions for locating, constructing, and maintaining on-site wastewater treatment systems.Water quality degradation influenced by diffuse sources at large watershed scales has been difficult to describe. Human modifications of natural landscapes can permanently alter hydrologic cycles and affect water quality (1, 2). Deforestation (3) and increased impervious surface area (4) have been linked with decreased infiltration and thus increased surface runoff. Overland flows concentrate pollutants and rapidly transport them down gradient where they eventually enter surface water systems and affect water quality (5, 6). A number of models have been developed to calculate overland and surface water flows (7, 8) and nutrient/chemical transport (9), but few studies have focused on microbial movement from land to water, particularly nontraditional fecal indicator bacteria that can be used to track human sources of pollution.Microbial contamination poses one of the greatest health risks to swimming areas, drinking water intakes, and fishing/shellfish harvesting zones where human exposures are highest (10–12). These highly visible areas often receive more attention than sources of contamination because identifying the origin of pollution in complex watersheds requires costly comprehensive investigation of environmental and hydrologic conditions across temporal and spatial scales (13). Grayson et al. (14) suggest using a “snapshot” approach that captures water quality characteristics at a single point in time across broad areas to provide information frequently missed during routine monitoring. Compared with long-term comprehensive investigations, the snapshot approach reduces the number of samples, cost, and personnel required to examine pollution sources.Escherichia coli concentrations are commonly used to describe the relative human health risk during water quality monitoring in lieu of pathogen detection. Studies attempting to trace pollution in water back to a specific land use with E. coli have rarely produced definitive conclusions (15, 16). Using molecular approaches, specific source targets can be isolated in complex systems and have recently been used to investigate land use and water quality impairments (17). Furtula et al. (18) demonstrated ruminant, pig, and dog fecal contamination in an agriculturally dominated watershed (Canada) using Bacteroides markers. The Bacteroides thetaiotaomicron α-1–6 mannanase (B. theta) gene has a high human specificity (19–22), but no studies to date have linked its presence to land use patterns.Reference conditions have been established for minimally disturbed environments based on measurements of macroinvertebrates, fish, and diatoms (23–25), but microbial reference conditions have not been adequately explored or defined. Based on 15 unimpaired California streams, microbial reference conditions for E. coli [1.0 log₁₀ most probably number (MPN)⋅100 mL⁻¹] and enterococci (1.2 log₁₀ MPN⋅100 mL⁻¹) were defined as being below state water quality thresholds (26). In the Great Lakes, a human health threshold of 2.37 log₁₀ E. coli MPN⋅100 mL⁻¹ (27), or a level equally protective of human health, has been adopted by all state governments. However, this health-associated reference level was derived from epidemiological studies undertaken at beaches throughout the United States (28, 29) with limited knowledge of local implications.In response to water quality degradation from human stressors and the poorly understood microbial conditions in large-scale fresh water systems such as the Great Lakes basin, this paper aims to (i) examine the spatial distribution of E. coli and a human specific source marker (B. theta) in 64 river systems that drain most of the state’s Lower Peninsula under baseflow conditions, (ii) identify baseflow reference levels of fecal contamination in rivers, and (iii) determine how key chemical, physical, environmental, hydrologic, and land use variables are linked to river water quality at large scales.