Streptococcus mutans and dental caries in humans: a bacteriological and immunological study

Plaque samples from caries-active subjects showed a higher incidence of S. mutans than plaque samples from caries-free subjects. This was especially evident in approximal incisor plaque. S. mutans serotype d was almost exclusively present in approximal plaque obtained from caries-active subjects. Tooth surfaces infected with S. mutans still harbored this micro-organism 10 months later, while uninfected tooth surfaces remained free of S. mutans.Caries development predominantly occurs on those tooth surfaces which harbor relatively high percentages of S. mutans (> 5%). It is unlikely that serum or saliva antibodies against S. mutans play a major role in the protection against dental caries in these caries-free subjects since subjects with the greatest number of decayed surfaces showed the highest antibody titre as measured by haemagglutination or by the enzyme-linked immuno sorbent assay (ELISA).The present investigation shows that serum or salivary antibodies against S. mutans do not seem to play a role in caries resistance in young adults. However, this does not preclude a role of antibodies, prior to infection with S. mutans, in man.     

Isolation and Characterization of Chloroplast DNA from the Duckweed Spirodela oligorrhiza

Chloroplast DNA of the duckweed Spirodela oligorrhiza, isolated by CsCl gradient centrifugation, was characterized by its buoyant density, guanine + cytosine content, melting behavior, circularity, and contour length. In all these characteristics, chloroplast DNA of S. oligorrhiza is similar to the chloroplast genomes of other higher plants, except that it has a significantly larger size.     

Production of Monoclonal Antibodies Specific for the i and 1,2 Flagellar Antigens of Salmonella typhimurium and Characterization of Their Respective Epitopes

Salmonella typhimurium expresses two antigenically distinct flagellins, each containing a different H antigen (i and 1,2), the combination of which is highly specific for this serotype. In this study, overlapping recombinant flagellin fragments were constructed from the fliC (H:i) and fljB (H:1,2) flagellin genes, and the expression products were tested for binding to H antigen-specific monoclonal and polyclonal antibodies. A minimal area, 86 amino acids for H:i and 102 amino acids for H:1,2, located in the central variable domain of each flagellin was required for the binding of serotype-specific antibodies, providing further evidence for the presence of a discontinuous H epitope. Two peptides comprising these areas were shown to be highly suitable for application as antigens in an enzyme-linked immunosorbent assay detecting S. typhimurium-specific antibody.     

Purification and characterization of a complex-bound and a free β-1,4-endoxylanase from the culture fluid of the anaerobic fungus Piromyces sp. strain E2

Two extracellular xylanases were purified to homogeneity from the culture filtrate of the anaerobic fungus Piromyces sp. strain E2 and their properties were studied. The enzymes are present in a High Molecular Mass complex (HMM-complex) and as free protein in nearly equal amounts. Both enzymes are most likely identical as all biochemical characteristics were identical. The molecular masses of the enzymes are 12.5 kDa, as estimated by gel chromatography and electrophoretic mobility. The activities of both enzymes are optimal at pH 6.0 and 50°C and the enzymes are stable up to 72h at 40°C. The enzymes have a pI of 9.1. The K _m and V _max, determined with xylan from oat spelts, were 3 mg · ml^-1 and 2600 IU · mg^-1 protein. The enzymes are active both on soluble and insoluble oat spelt xylan. The purified xylanases are inactive against Avicel, carboxymethylcellulose, p-nitrophenyl--d-glucoside, and p-nitrophenyl--d-xyloside. The products of the pure enzymes are predominantly xylo-oligosaccharides, indicating that the enzymes act as endoxylanases (1,4--d-xylan xylanohydrolases, EC 3.2.1.8).     

Digoxigenin-labelled inv- and ail-probes for the detection and identification of pathogenic Yersinia enterocolitica in clinical specimens and naturally contaminated pig samples

A non-radioactive colony hybridization method was developed for the rapid detection of Yersinia enterocolitica in primary isolates and for differentiation between pathogenic and non-pathogenic strains. The method is based on, respectively, the presence of the inv -locus in all Yersinia spp. and the presence of the ail -gene in pathogenic Y. enterocolitica only. Hybridization results with ail -probes of 132 strains of Y. enterocolitica were in good agreement with pathogenicity phenotypes as indicated by a tissue culture invasion (TCI) assay and by serotyping. All TCI⁺ strains and only two TCI^- strains were positive by hybridization with ail. Hybridization results with inv - or ail -probes of 150 primary isolates of human, animal or slaughterhouse origin were compared with those of conventional methods to detect and identify Y. enterocolitica. All samples that were positive for Yersinia spp. by cultivation (four of 66) or were positive for pathogenic Y. enterocolitica by cultivation and serotyping (six of 84) were also positive by hybridization with, respectively, the inv - or ail -probe. In three slaughterhouse swab samples, in which Yersinia spp. were not detected by cultivation (2%), strong positive hybridization signals were obtained with the inv - and/or ail -probe. Four other swab samples which were negative by cultivation produced weak positive signals by hybridization with inv - and/or ail - probes. These results indicate that the method can be used for (1) the identification of pathogenic Y. enterocolitica isolates and (2) the detection of Yersinia spp. in primary isolates of naturally contaminated samples.     

Antimicrobial activity of lactobacilli: preliminary characterization and optimization of production of acidocin B, a novel bacteriocin produced by Lactobacillus acidophilus M46

Approximately 1000 lactobacillus strains were isolated and screened for the production of antimicrobial activity, using a target panel of spoilage organisms and pathogens. Only eight positive strains were found; two of these were studied in more detail. Lactobacillus salivarius M7 produces the new broad spectrum bacteriocin salivaricin B which inhibits the growth of Listeria monocytogenes, Bacillus cereus, Brochothrix thermosphacta, Enterococcus faecalis and many lactobacilli. A new atypical bacteriocin produced by Lact. acidophilus M46, acidocin B, combines the inhibition of Clostridium sporogenes with a very narrow activity spectrum within the genus Lactobacillus and was selected for further characterization. Acidocin B is sensitive to trypsin, heat-stable (80°C for 20 min) and can be extracted from the culture supernatant fluid with butanol. Native acidocin B occurs as a large molecular weight complex (100 kDa), while with SDS-PAGE the partly purified activity migrates as a peptide of 2·4 kDa. Optimization of the cultivation conditions resulted in an eightfold increase of the amount of acidocin B produced during growth. Growth is not necessary for acidocin B production; washed producer cells can synthesize the bacteriocin in a chemically defined production medium. The application potential of acidocin B is discussed.     

Apolipoprotein A-I from striped bass (Morone saxatilis) demonstrates antibacterial activity in vitro

HDL and apolipoprotein A-I from teleostean fishes demonstrate in vitro activity against gram-positive and gram-negative bacteria. In this study, we purified ApoA-1 from striped bass (Morone saxatilis) plasma and examined its in vitro antibacterial activity against Streptococcus sp., Escherichia coli, and Mycobacterium marinum. In addition, we obtained sequence for a putative striped bass ApoA-1 gene, which when translated contained the identical sequence generated from N-terminal sequencing of the purified ApoA-1. The predicted secondary and tertiary structures contained the characteristic proline residues and high alpha-helical content conserved between mammals and fishes. Purified ApoA-1 exhibited antibacterial activity against the bacteria assayed. Concentrations of 125 microg/mL for E. coli, 250 microg/mL for Streptococcus sp., and 250 microg/mL for M. marinum, inhibited bacterial growth by 50% compared to control. ApoA-1 plasma concentrations in experimental and wild fish ranged from undetectable levels to greater than 5 mg/mL, indicating that striped bass ApoA-1 is an effective antibacterial agent at concentrations below the range of physiological concentrations in striped bass plasma. We therefore conclude that ApoA-1 could play a role in innate defense against bacterial pathogens in striped bass.     

Simulation of the mechanical strength of a single collagen molecule

We perform atomistic simulations on a single collagen molecule to determine its intrinsic molecular strength. A tensile pull simulation to determine the tensile strength and Young's modulus is performed, and a simulation that separates two of the three helices of collagen examines the internal strength of the molecule. The magnitude of the calculated tensile forces is consistent with the strong forces of bond stretching and angle bending that are involved in the tensile deformation. The triple helix unwinds with increasing tensile force. Pulling apart the triple helix has a smaller, oscillatory force. The oscillations are due to the sequential separation of the hydrogen-bonded helices. The force rises due to reorienting the residues in the direction of the separation force. The force drop occurs once the hydrogen bond between residues on different helices break and the residues separate.     

Heat resistance of Bacillus cereus emetic toxin, cereulide

Aims: The study describes the effects of heating temperature and exposure time on the thermal stability of cereulide under different conditions (pH, presence/absence of oil phase and cereulide concentration). Methods and Results: Cereulide heat inactivation was investigated at 100, 121 and 150°C under different alkaline pH values (8.6–10.6) and in the presence of oil phase (0.6–1.4%). Three different cereulide concentrations (0.5, 5 and 6 μg ml^?1) were used. Cereulide detection was performed with computer‐aided semen analyzer and with HPLC–MS. Highly alkaline pH was needed to achieve inactivation. At lower cereulide concentrations less drastic conditions were needed. Removal of alkaline buffer after the heat treatment resulted in the recovery of toxic activity. Conclusions: Heat stability of cereulide has been proved to be remarkable, even at highly alkaline pH values, at all temperatures tested. The loss of activity appeared to be reversible. Significance and Impact of the Study: The study demonstrates the inability of any heat treatment used in the food industry to inactivate cereulide. Food safety has to rely on prevention and cold chain maintenance. Cleaning practices also need to be adapted as cereulide may remain in its active form upon sterilization of used material.     

In vitro response of the striped bass natural resistance-associated macrophage protein, Nramp, to LPS and Mycobacterium marinum exposure

Mycobacteriosis in Chesapeake Bay (USA) striped bass Morone saxatilis is an ongoing disease problem with important economic implications for a large commercial and recreational fishery. Additionally, striped bass serve as a reservoir of potential mycobacterial zoonoses. Recently, we described a striped bass gene homolog of the natural resistance-associated macrophage protein family (MsNramp), which is responsible for resistance to mycobacterial infections in mice. Striped bass MsNramp is strongly induced in peritoneal exudate cells (PE) in vivo after intraperitoneal injection with Mycobacterium spp. The purpose of the present study was to investigate short-term in vitro MsNramp expression and reactive oxygen intermediate (ROI) production in primary cultures of adherent PE after exposure to bacterial lipopolysaccharide (LPS), or live- or heat-killed (HK) Mycobacterium marinum. PE expressed significantly higher levels of MsNramp at 4 and 24 h post-treatment with live and HK M. marinum. MsNramp response to LPS was dose-dependent in these cells, with maximum expression at 4 h and 20 microg/ml LPS. Treatment of PE with LPS resulted in increased intracellular superoxide anion levels, whereas treatment with live M. marinum caused a significant depression. This study is the first report of induction of a teleost Nramp in vitro by mycobacteria, and supports findings of teleost Nramp induction by LPS.