Showing papers in "Microbial Ecology in Health and Disease in 1989"

Production of a Broad Spectrum Antimicrobial Substance by Lactobacillus reuteri

Abstract: Lactobacillus reuteri resides in the gastrointestinal tract of humans, swine, poultry and other animals. Resting cells of this species convert glycerol into a potent, broad-spectrum antimicrobial substance termed reuterin. Reuterin is a low molecular weight, neutral, water soluble compound, capable of inhibiting growth of species representing all bacterial genera tested thus far, including: Escherichia, Salmonella, Shigella, Proteus, Pseudomonas, Clostridium and Staphylococcus. Also affected, but to a lesser degree, are lactic acid bacteria belonging to the genera Streptococcus, Pediococcus, Leuconostoc, and Lactobacillus. In this report we describe a method to screen lactobacilli for production of unique antimicrobial substances and the discovery of reuterin.

Effect of Probiotic Supplements of Lactobacillus acidophilus and Bifidobacterium adolescentis 2204 on β-glueosidase and β-glueuronidase Activity in the Lower Gut of Rats Associated with a Human Faecal Flora

Abstract: Rats associated with a human faecal flora were dosed for 3 d with supplements of live Lactobacillus acidophilus NCFM or Bifidobacterium adolescentis 2204. The treatments suppressed the β-glucosidase and β-glucuronidase activities in the colon. The effect was only statistically significant for the L. acidophilus treated rats.

Development of Periodontal Microflora on Human Serum

Abstract: This study was undertaken to identify the ecological niches of members of subgingival microflora. The succession of species during enrichment batch growth of subgingival plaque organisms in serum was monitored. Three phases could be distinguished during growth, firstly carbohydrate consumption by rapidly growing saccharolytic bacteria such as Eubacterium saburreum or Bifidobacterium adolescentis and Streptococcus spp. leading to lactic and formic acid production. Secondly a later phase in which protein was hydrolysed, some amino acid fermentation took place, remaining carbohydrates were used and lactate and formate were consumed; growth was dominated by Bacteroides intermedius or Bacteroides oralis , Veillonella parvula , Eubacterium spp. and Fusobacterium nucleatum , and a final phase characterised by progressive protein degradation and extensive amino acid fermentation by the predominant species, Peptostreptococcus micros and Eubacterium brachy . Toxic products such as sulphide, ammonia, butyric acid and other fatty acids accumulated. Keywords: Peptostreptococcus micros ; Eubacterium ; Bacteroides ; Fusobacterium nucleatum ; Periodontitis; Serum degradation; Niche; Growth-curve.

The Role of Surface Free Energy in the Early In vivo Formation of Dental Plaque on Human Enamel and Polymeric Substrata

Abstract: Strips of teflon and cellulose acetate were glued to the upper lateral incisors of human volunteers in a split mouth, double blind study on the influence of the substratum surface free energy (s.f.e.) on supragingival dental plaque accumulation during a three day period of no oral hygiene. Plaque accumulation, microbial composition of the plaque and s.f.e. of the microorganisms were determined and compared to plaque developed on natural enamel surfaces. Significantly less microorganisms colonised the polymer surfaces (p < 0.002). Streptococcus sanguis I was the predominant microorganism found in enamel samples, comprising about one-third of the total microflora, whereas it was recovered infrequently and in lower numbers from the polymeric surfaces, which predominantly contained Streptococcus sanguis II. Only on cellulose acetate sometimes high numbers of Streptococcus mitis and Streptococcus morbillorum were detected. The mean s.f.e. of the total plaque flora was lowest on teflon (84.5 mJ m -2 ) followed by cellulose acetate (86.0 mJ m -2 ), whereas enamel harboured a microflora with a significantly higher mean s.f.e. (93.0 mJ m -2 ; p<0.05). Also within the same bacterial species lower s.f.e. strains were isolated from the polymer surfaces compared to enamel. The results conform to a previously postulated model in which the interfacial free energy is the driving force for adhesion of microorganisms to solid surfaces. Keywords: Surface free energy; Bioadhesion; Oral microflora; Bacteria; Foreign Bodies.

An In vitro Study of Ileal Colonisation Resistance to Escherichia coli Strain Bd 1107/75 08 (K88) in Relation to Indigenous Squamous Gastric Colonisation in Piglets of Varying Ages

Abstract: Ileal colonisation resistance to Escherichia coli strain Bd 1107/75 08 (K88) in piglets aged 5, 26 and 47 days and the possible relation to indigenous gastric colonisation was studied. In diluted ileal extracts, heat labile growth inhibitory activity was detected. At 26 d, the in vitro adhesion of K88 fimbriae on E. coli to ileal mucus was markedly increased, while the level of in vitro adhesion of Lactobacillus crispatus strain 152 to stomach tissue pieces decreased. In addition, scanning electron microscopy revealed that the composition of the squamous stomach epithelium associated flora changed dramatically at this time. From these results, it appears that age and consequently related factors influence the ileal colonisation resistance to E. coli strain Bd 1107/75 08 (K88) and the composition of the squamous indigenous gastric flora. Keywords: E. coli K88; Lactobacillus; Colonisation resistance; Adhesion; Growth.

Inhibition of Shigella sonnei and Enterotoxigenic Escherichia coli by Volatile Fatty Acids in Mice

Abstract: The role of volatile fatty acids (VFA) in resistance against the colonisation of the intestinal tract ofmice with two enteric pathogens, Shigella sonnei 3SR and enterotoxigenic E. coli 2SR (ETEC 2SR) was determined. The implantation dose 50 (ID50) of S. sonnei 3SR strain for untreated mice was greater than 5.0 x 10 9 viable cells and was 1.8 x 10 6 viable cells for ETEC 2SR. Administration of streptomycin to the mice prior to challenge lowered the ID50 to fewer than one viable cell for both organisms. The antibiotic caused an increase in the pH of caecal contents from 6.66 in untreated animals to 6.94 in treated animals and a decrease in total VFA concentrations from 109.11 microequivalent per ml in untreated animals to 78.31 microequivalent per ml in treated animals. The discontinuation of streptomycin treatment resulted in a gradual restoration of colonisation resistance accompanied by an increase in total VFA concentration and a decrease in pH of caecal contents. These values returned to pre-treatment levels by day 7. VFA were added to nutrient broth in concentrations present in the caeca of either untreated or streptomycin treated mice and the pH levels were adjusted accordingly. S. sonnei 3SR multiplied in broth adjusted to simulate conditions present in the caeca of treated mice but failed to multiply in broth adjusted to simulate conditions present in the caeca of untreated mice. ETEC 2SR, on the other hand, multiplied in both types of broth but its total populations after 24 h incubation were significantly smaller in broth adjusted to simulate conditions present in the caeca of untreated mice. The results demonstrate that the multiplication of both pathogens is inhibited by the presence of VFA. They indicate that VFA operating at the pH level present in the intestinal tract of conventional mice represent important factors responsible for resistance against colonisation with S. sonnei and ETEC. Keywords: Shigella sonnei ; Enterotoxigenic E. coli ; Volatile fatty acids; Mice.

Colonisation index of the oral cavity: a novel technique for monitoring colonisation defence

Abstract: The constancy of oropharyngeal flora in healthy people is based on intact defence against colonisation. Conditions which can interfere with this defence are underlying disease and medical interventions, and may be associated with alterations in the oropharyngeal flora. The oral flora was assessed by means of a modified oral rinse method based on pre-enrichment in nutrient medium. The colonisation index of the oral cavity was defined as the sum of the log 10 of the concentrations of a particular microorganism isolated from one ml of oropharyngeal washing specimens, divided by the number of oral washings. This index was evaluated as an indicator of (decreased) colonisation defence by comparing a group of healthy volunteers with a group of post-surgery head and neck oncology patients. Oral colonisation indices for Enterobacteriaceae , Pseudomonadaceae and Acinetobacter spp. as well as for Staphylococcus epidermidis were statistically significantly higher in the patient group compared to the volunteers. This clear reduction in colonisation defence, resulting in a high carriage rate of potentially pathogenic Gram-negative bacilli, may be a result of the combination of underlying disease (cancer) and surgical intervention (debulking or radical resection of tumor). Keywords: Oropharynx; Colonisation index; Colonisation defence; Oral rinse method; Head and neck cancer

The Ecology of Bacteriocin-producing Strains of Streptococcus salivarius

Abstract: Interest in bacteriocin-producing components of the human normal oral microbiota centres on their possible interference with colonisation by potentially pathogenic bacteria. Certain strains of Streptococcus salivarius produce bacteriocin-like agents displaying exceptional inhibitory activity toward Lancefield Group A streptococci. Four individuals were identified as naturally harbouring high proportions (> 90 per cent) of bacteriocin-producing strains of S. salivarius . Bacteriocinogenic isolates from three of these individuals and the previously described strain 20P3 displayed identical patterns of inhibition when tested by a deferred antagonism method against a series of ten indicator organisms. The fourth individual harboured S. salivarius strains producing a distinct, though possibly related, inhibitory pattern. All agents were of apparently low molecular weight (< 8000) and were stable to heating at 80°C for 30 min. The four individuals harbouring bacteriocinogenic S. salivarius strains possessed significantly greater proportions (p < 0.001) of bacteriocin-resistant Gram-positive alpha-haemolytic cocci than did 13 control individuals (not found to harbour bacteriocinogenic S. salivarius strains). These results accord with the idea that the production of active bacteriocins in situ should select a relatively bacteriocin-resistant accompanying microbiota. Keywords: Streptococcus salivarius ; Bacteriocin; Microbial interference; Indigenous microbiota; Colonisation resistance.

Adaptive Changes in a Strain of Streptococcus mutans during Colonisation of the Human Oral Cavity

Abstract: When a bacterial strain undergoes a change in environment, its ability to colonise a new niche is optimised over time by the process of natural selection. In this study, a readily identifiable strain of Streptococcus mutans was recovered from supragingival dental plaque of human subjects 3 y after implantation. Fifty fresh isolates were compared to the original laboratory strain that was used to infect these subjects. It was found that the fresh isolates adhered less well than the laboratory strain to saliva-coated hydroxyapatite. The fresh isolates also produced more acetoin and less lactic acid, and had a greater cell yield during aerobic growth in glucose-containing medium. During growth in the presence of sucrose, the fresh isolates produced slightly greater amounts of plaque than the laboratory strain. No differences were observed between fresh and laboratory adapted strains with regard to bacteriocin production, antigenic composition, nutrient requirements, growth rates, or resistance to tetracycline. These results indicate that environmental factors operating in the oral cavity of human subjects select variants of S. mutans that are better adapted to proliferate in vivo . Identification of these factors is important to our general understanding of the ecology of S. mutans and to the development of therapeutic methods. Keywords: Streptococcus mutans ; Adaptation; Implantation.

The Distribution of Hydrolytic Enzymes Among Gram-negative Bacteria Associated with Periodontitis

Abstract: The ability of representative oral microorganisms to degrade biopolymers, such as collagen, chondroitin-4-sulphate, hyaluronic acid, heparin and proteins was investigated using both native and synthetic substrates. Porphyromonas gingivalis and Bacteroides heparinolyticus were the only species which had heparinase activity. Hydrolysis of hyaluronic acid and chondroitin-4-sulphate was detected in P. gingivalis irrespective of the method used. Both enzymes were largely cell associated and increased up to 120 h. Hyaluronic acid and chondroitin-4-sulphate degradation was detected in B. melaninogenicus, B. oralis, Fusobacterium nucleatum, Capnocytophaga ochracea and Veillonella parvula by a spectrophotometric assay but not by a plate method. Collagenase activity was mainly associated with P. gingivalis ; activity was largely extracellular and reached a maximum after 96 h. Several other species such as the black-pigmented bacteroides, F. nucleatum and C. ochracea showed very weak activity. All species tested except B. oralis possessed some proteolytic activity against gelatin, casein and skimmed milk. Fluorometnc and chromogenic substrates which contained an arginine residue were readily hydrolysed. Keywords: Oral flora; Hydrolytic enzymes; Periodontal disease; Gram-negative anaerobes.

Characterisation of Protective Antibodies in Hamsters Immunised Against Clostridium difficile Toxins A and B

Abstract: Toxigenic Clostridium difficile is the major cause of antimicrobial agent-associated pseudomembranous colitis in humans and of ileocaecitis in golden Syrian hamsters. The pathogenicity of C. difficile is believed to be dependent on the production of two immunologically and biochemically distinct toxins: toxin A (enterotoxin) and toxin B (cytotoxin). In earlier investigations we demonstrated that hamsters immunised against toxins A and B are protected against clindamycin-induced C. difficile -associated ileocaecitis and that this protection is transferred to infant hamsters through maternal milk. In the present investigation, the class-specific immunoglobulin response in adult and infant hamsters immunised against C. difficile toxins A and B was examined using enzyme linked immunosorbent assays. Parenteral immunisation of adult hamsters against toxins A and B induced IgG, IgA and IgM antibodies in hamster sera specific for these two toxins. IgG antibodies to toxins A and B predominated in the milk and intestines of adult hamsters immunised against toxins A and B and in the sera and intestine of infants from immunised adult hamsters. No significant IgA or IgM antibodies to toxins A and B were detected in maternal milk and intestines or in infant sera and intestines. IgG antibodies to toxins A and B were detected in infant caecal contents up until 10 d and in sera up until approximately 24 d of age. The results from this investigation suggest that serum derived IgG antibody to C. difficile toxins is primarily responsible for protection of adult and infant hamsters against C. difficile -associated ileocaecitis when immunised parenterally against toxins A and B. Keywords: Clostridium difficile ; Toxin A; Toxin B: Antibody; Immunisation; Hamster; Ileocaecitis; Enterotoxin; Cytotoxin.

Growth of Oral Microflora on Saliva from Different Glands

Abstract: In this study we have investigated the growth of batch-wise enrichment cultures of supragingival plaque on parotid, (PAR) submandibular-sublingual (SM-SL) and clarified whole (CHW) saliva from one volunteer, who also donated the plaque sample All three salivas supported rapid growth of a mixed oral microflora Salivary glycoproteins were completely deglycosylated during growth, while most proteins were left intact The enrichment cultures on PAR and SM-SL salivas had different microbial compositions B oralis was dominant in the culture on PAR saliva, but absent in the culture on SM-SL saliva In contrast E lentum was found in a high proportion in SM-SL saliva, but was absent in the culture on PAR saliva S mitis was a dominant organism in both cultures, but the isolates from PAR and SM-SL saliva belonged to different biotypes The enrichment on CHW saliva appeared to have an intermediate composition and most species and biotypes isolated either from the culture on PAR or from SM-SL saliva were found in CHW saliva The various glycosidase and peptidase activities produced in the enrichment were largely cell-associated and found to be the same as those normally found in saliva and dental plaque in vivo Taken together the results support the contention that the normal oral microflora maintains itself in the oral cavity by using salivary glycoproteins as a substrate Moreover saliva seems to act as a selective force in the oral cavity, since different types of glycoproteins as found in PAR and SM-SL saliva supported the growth of different microfloras Keywords: Saliva; Glycoproteins; Glycosidases; Peptidases; Oral microflora; Streptococci; Bacteroides; Adhesion

Bacteroides gingivalis: Recognition of a 47 kDa Outer Membrane Protein by Sera from Patients with Chronic Periodontitis

Abstract: Sarcosyl-insoluble outer membrane components of Bacteroides gingivalis were separated by SDS-PAGE and electroblotted onto nitrocellulose membranes. They were then probed with sera from patients with a history of advanced periodontitis (CPITN of 4) and from a control group of subjects with little or no history of periodontitis (CPITN of 3 or less). A47 kDa antigen was recognised by sera fromall fifteen patients but not by nine often subjectsin thecontrol group. Keywords: Bacteroides gingivalis ; Outer membrane; Periodontal disease.

A Morphological Study of the in situ Tissue-Associated Autochthonous Microflora of the Human Vagina

Abstract: Direct examination of scrapings from three locations in the vaginas of healthy volunteers, at different times of the menstrual cycle, revealed the presence in situ of tissue-associated autochthonous bacterial populations. Most of the bacteria in these scraped samples were intimately associated with the surfaces of epithelial cells, to which they were connected by elements of their exopolysaccharide glycocalyces. Washing of the epithelial sites, before sampling, removed vaginal secretions and their associated bacterial populations but did not remove the tissue-adherent bacterial microcolonies and individual cells. When epithelial cells were exposed to the high shear forces of vortex mixing, centrifugation and sonication, this firmly adherent bacterial population was almost entirely retained at the vaginal cell surface. The nature and strength of this intimate bacteria-tissue association may be important in the colonisation resistance afforded by autochthonous bacteria and in the pathogenesis of vaginal infections, and as such should be a major consideration in future ecological studies. Keywords: Human vagina; Autochthonous flora; Tissue-association; Ecology.

Immunoglobulin G Cleaving Species in Serum-degrading Consortia of Periodontal Bacteria

Abstract: Previous experiments have indicated that the enrichment of subgingival plaque in human serum led to specific consortia of micro-organisms. The present study was undertaken to identify the role which particular organisms have in these consortia in serum degradation. Firstly, isolates from consortia were tested for their ability to degrade immunoglobulin G, the major glycoprotein in serum. From the variety of species tested, only Bacteroides intermedius , Bacteroides oralis and Eubacterium nodatum were capable of IgG-cleavage and protein-core attack. Reconstitutions of consortia showed that without 'cleavers', combinations of saccharolytic specialists such as Bifidobacterium adolescentis and Streptococcus sanguis II and/or of (poly-)peptide- and amino acid-fermenting specialists such as Peptostreptococcus micros , Eubacterium brachy and Fusobacterium nucleatum were incapable of extensive growth on serum and of immunoglobulin A and G consumption. The protein cleaving organisms make glycoproteins accessible as substrates to other members of these consortia. Furthermore, these IgG-cleavers may also enhance growth of others by eliminating host-defence mechanisms in vivo . These consortia seem well adapted to their sites of isolation from various anaerobic body infections. Keywords: Immunoglobulin G; Periodontitis; Serum degradation; Bacteroides intermedius ; Bacteroides oralis ; Eubacterium nodatum ; Peptostreptococcus micros.

Enumeration of Intestinal Bifidobacteria by Growth on a Semi-selective Medium and GLC Assay of Acetic Acid Production

Abstract: A method is described for the enumeration of bifidobacteria in faeces which consists of a dilution count on a semi-selective medium followed by assay of the amount of acetic acid produced under standard conditions. With one exception ( Lactobacillus reuteri ) the lactobacilli always produced less acetic acid than did the bifidobacteria. L. reuteri could be distinguished from the bifidobacteria by the high concentration of acetaldehyde which it produced. Keywords: Bifidobacteria identification; Gut flora; Acetate production

The in vivo Effect of Dietary Buffering Capacity on the Bifidogenic Activity of Human Milk Oligosaccharides

Abstract: The buffering capacity of a milk diet is known to be involved in the control of a newborn's bifidobacterial flora. Other factors could modulate the bifidobacterial flora e.g. bifidus factors present in human milk, which are growth-promoting for Bifidobacterium bifidum. The aim of this study was to investigate in gnotobiotic mice the effects of human milk bifidus factors upon colonisation by bifidobacteria according to the dietary buffering capacity. A high buffering capacity diet encouraged the implantation of higher numbers of bifidobacteria in monoxenic mice than a low buffering capacity one. However, supplementary feeding with human milk oligosaccharides (gynolactose) did not support the proliferation of B. bifidum , although the gynolactose bifidus factors were not destroyed during digestion. Furthermore the addition of gynolactose to a high buffering capacity diet led to the breakdown of some of the intestinal glycoproteins detected in the bifidogenic extract of stools from germfree mice. In addition, although the human milk oligosaccharides stimulated the excretion of intestinal bifidus factors when supplementing a low buffering capacity diet, they failed to do so on a high buffering capacity diet. These results suggest that the ability of human milk oligosaccharides to encourage the proliferation of B. bifidum depends upon their capacity to stimulate intestinal bifidus factors. This capacity seemed to be related to the buffering capacity of the diet. Keywords: Bifidobacterium bifidum; Gynolactose; Germ free mice: Intestinal mucus.

Susceptibility of Clostridium difficile Toxins A and B to Trypsin and Chymotrypsin

Abstract: Previous studies describing the inactivation of toxins A and B of Clostridium difficile by trypsin and chymotrypsin used impure toxin and protease preparations. We repeated these studies using highly purified toxin, and trypsin and chymotrypsin that were not contaminated with each other. Our findings show that toxin A is resistant to trypsin but not chymotrypsin and that toxin B is inactivated by both proteases. Neither toxin was activated by the proteases. Keywords: C. difficile ; Toxin A; Toxin B; Proteases.

Binding of Immunoglobulins and other Proteins to Bacteroides gingivalis

Abstract: Bacteroides gingivalis bound IgG labelled with three different enzymes even in well-buffered isotonic systems. This nonimmunological binding interferes with enzyme linked immunosorbent assay (ELISA) of salivary antibodies against B. gingivalis . B. melaninogenicus and B. intermedius showed only weak IgG binding while nine other species of oral bacteria did not bind IgG. The broad specificity of the receptor binding IgG to B. gingivalis includes not only IgG and IgA, but also proteins unrelated to immunoglobulins. Like the haemagglutinin of B. gingivalis , the receptor that binds IgG has a specificity for arginine residues with an adjacent or neighbouring aromatic amino acid residue. In contrast to the strong IgG binding of its parent, an avirulent variant B. gingivalis W50/BE1 that is deficient in trypsin-like activity, has a low level of IgG binding. Our results suggest firstly either an inactive or deactivated form of a trypsin- or papain-like enzyme, or a molecule such as the haemagglutinin, binds immunoglobulins and other proteins to the surface of B. gingivalis . Secondly, binding to arginine in proteins is an important ecological factor for B. gingivalis since it is a property shared by its IgG binding receptor, its haemagglutinin and its trypsin- or papain-like enzyme. Finally, the sensitivity of ELISA to non-immunological reactions necessitates establishment of the specificity of immunoassays at levels of sensitivity comparable to those of the assays themselves. Keywords: Bacteroides gingivalis ; ELISA; Protein receptor; Immunoglobulins; Arginine specificity.

Campylobacter pylori in Relation to Other Aerobic and Anaerobic Microorganisms in Patients with Gastric Diseases

Abstract: The degree of Campylobacter pylori colonisation in relation to other aerobic and anaerobic microorganisms on antral mucosal biopsies was investigated in 53 patients with dyspeptic symptoms. Microbial colonisation was found in 94 per cent of the patients. Streptococci and Campylobacter pylori were the most common findings. Microorganisms from the normal oropharyngeal flora, aerobic Gram-negative rods and yeasts were found irrespective of the diagnosis, and even in subjects with normal mucosa. C. pylori was with one exception only found in patients with gastritis. All patients with peptic ulcer had gastritis and 83 per cent of these harboured C. pylori . When active chronic gastritis was present, C.pylori was isolated in 85 per cent of the cases. When present, C. pylori was always found in higher numbers than other microorganisms. No relation between the gastric pH and the microbial colonisation of the mucosa was found. Keywords: Campylobacter pylori ; Colonising microorganisms; Gastritis; Peptic ulcer.

The Relationship between Coaggregation Properties and Surface Structures of Bacteroides intermedius

Abstract: Seven out of 20 Bacteroides intermedius isolates coaggregated with two Actinomyces viscosus and four A. naeslundii strains. None coaggregated with seven other strains representing five Gram-positive oral species. Coaggregating ability was unrelated to B. intermedius serogroup, surface hydrophobicity, or morphology of fibrils or ruthenium red (RR) staining layers. The coaggregation adhesin on B. interrnedius isolates recognising A. viscosus and A. naeslundii was unaffected by serum, saliva, sugars and Congo red. It was inactivated by NaIO 4 , heat, proteinase K and, in 4/7 isolates, by trypsin but not by heat-inactivated enzymes. Coaggregation adhesins on serogroup I isolates were resistant to trypsin, and were less susceptible than those on other strains to heat and proteinase K. Cell surface hydrophobicity was unaltered by NaIO 4 and trypsin. RR staining matrices and capsules (detected by staining with Indian ink) were unaffected by trypsin, but NaIO 4 removed both. Capsules and fibrils were removed at different times by NaIO 4 and were therefore different structures. Coaggregating ability disappeared with removal of fibrils indicating the adhesin was associated with the fibrillar layer. All B. intermedius isolates carried two types of fibril. One was removed by trypsin treatment but was not involved in coaggregation. The other was not removed by trypsin or proteinase K. The proteinase K and heat-inactivatable coaggregation adhesin may be a non-structural component of these fibrils. Keywords: B. intermedius ; Coaggregation; Adhesins; Actiriomyces spp. ; Ultrastructure; Fibrils; Ruthenium red; Capsules; Hydrophobicity.

Inhibitory Effects of Volatile Fatty Acids and Low pH on the Multiplication of Enteric Pathogens In Vitro in Caecal Contents of Mice

Abstract: The inhibitory effect of volatile fatty acids (VFA) and low pH on the multiplication of Shigella sonnei 3SR and enterotoxigenic Escherichia coli 2SR (ETEC 2SR) in caecal contents of mice was determined. Multiplication rates and population sizes of both pathogens were greater in contents obtained from streptomycin treated mice at pH 6.94 containing 78.32 μeq total VFA per gram than in contents from untreated conventional mice at pH 6.66 containing 109.11 μeq total VFA per gram. When contents from streptomycin treated mice were adjusted to pH 6.66 and to contain 109.11 μeq total VFA per gram, values observed in contents from untreated mice, the multiplication rates and population sizes of both pathogens were lower in the adjusted contents than in unadjusted contents. Lowering the pH alone without VFA addition did not influence the multiplication of ETEC 2SR but caused a moderate decrease in the multiplication rate and population sizes of S. sonnei 3SR. These effects, however, were not as pronounced as when VFA were also added. Conversely, neutralisation of VFA present in caecal contents from untreated mice by increasing the pH from 6.66 to 7.50 reversed inhibition. Growth curves of both S. sonnei 3SR and ETEC 2SR in contents at high pH were almost identical to curves obtained when the organisms were multiplying in contents from streptomycin treated mice. The results provide strong evidence that the combined effects of high concentrations of VFA and low pH in caecal contents of conventional mice are important determinants of colonisation resistance against enteric pathogens in mice. Keywords: Volatile fatty acids; pH; Enteric pathogens; Caecal contents; Mice.

Influence of Antibiotics Administered in Therapeutic Doses on Colonisation Resistance Against Enteric Pathogens in Mice

Abstract: Colonisation resistance against oral challenge with Salmonella typhimurium, Shigella flexneri and enterotoxigenic Escherichia coli (ETEC) was determined using untreated mice and mice pre-treated with therapeutic doses of either amoxicillin, chloramphenicol, erythromycin, penicillin or trimethoprim-sulfamethoxazole (TMS), or with streptomycin, administered in high dose. The effects of these antibiotics on the pH levels and volatile fatty acid (VFA) concentrations of caecal contents were also determined. Only streptomycin treatment altered colonisation resistance. The incidence of caecal colonisation by all three pathogens and their populations were significantly greater in streptomycin treated than untreated mice. Except for an increase in the incidence of colonisation by ETEC after erythromycin treatment, none of the antibiotics, administered in therapeutic doses, produced significant effects on colonisation resistance. Relative to untreated mice, streptomycin treatment caused a statistically significant decrease in total caecal VFA's, from 60.80 μeq/g to 21.63 μeq/g, and a significant increase in pH from 6.75 to 7.04. Treatment with TMS caused an increase in total caecal VFA to 91.18 μeq/g. Treatment with the other antibiotics failed to produce significant changes in total caecal VFA's and, with the exception of chloramphenicol treatment resulting in a pH of 7, in caecal pH levels. All of the pathogens were sensitive to the presence of VFA in nutrient broth. In every case, their populations, after 12 h incubation, were greatest in broth containing 21.63 μeq/ml VFA adjusted to pH 7.04, simulating conditions in the caecum of streptomycin treated mice, and were smallest in broth containing 91.18 μeq/ml VFA adjusted to pH 6.74, simulating conditions in the caecum of TMS treated mice. Despite the suppressive effects of conditions simulating TMS treatment in vitro , mice treated with this combination of antibiotics were no more resistant to colonisation with the pathogens than were untreated mice. The results demonstrate that antibiotics, administered in therapeutic doses, have little effect on colonisation resistance against enteric pathogens in mice and suggest that a similar situation may exist in humans. Keywords: Antibiotics; Colonisation resistance; Mice.

A Comparison of Bacterial Growth Inhibiting Effects of Six Commercially Available Mouthrinses

Abstract: In this study the bacterial growth inhibiting effects of six commercially available mouthrinses (Hibident®, Prodent®, Merocet®, Listerine®, Veadent® and Mendol®) were determined. Hibident® was used as a positive control. Five strains were tested ( Streptococcus mutans C67, Streptococcus sanguis CH3, Veillonella alcalescens V1, Lactobacillus acidophilus JP and Actinomyces viscosus C74), as representatives of the supragingival human microflora. The Maximal Inhibiting Dilution (MID) was measured in batch cultures for each product and strain. With respect to the positive control, Hibident® (containing 0.2 per cent chlorhexidine), the most effective product was Meridol® (containing 125 ppm aminefluoride 297 and 125 ppm stannous fluoride) followed by Merocet® (containing 0.05 per cent cetylpyridinium chloride), Veadent® (containing 0.03 per cent sanguinarine), Listerine® (containing phenolic compounds) and Prodent® (containing 0.5 per cent sodium fluoride). Although all products have been separately reported to yield a plaque reduction in vivo , this study provides a firm basis for a comparison between products, as they were all evaluated in a similar way. Keywords: Mouthrinses; Bacterial growth; Maximal Inhibiting Dilution.

Adhesion of Non-coccal Dental Plaque Microorganisms to Buccal Epithelial Cells: Inhibition by Saliva and Amphipathic Agents

Abstract: In the present investigation, the in vitro adhesion of dispersed non-coccal dental plaque bacteria to buccal epithelial cells was examined. Human buccal epithelial cells, incubated in the presence of dispersed supragingival dental plaque, bound averages of ca. 6-15 non-coccal forms/epithelial cell, as opposed to indigenous levels of only 0.2-0.6 on buccal epithelial cells incubated in buffer alone. Adhesion was strongly inhibited by amphipathic agents (Tween 80, sodium dodecyl sulphate, and emulsan), and by saliva. The data suggests firstly that non-coccal bacteria can adhere to epithelial cells in vitro , but may be prevented from doing so in situ by saliva, and secondly that hydrophobic interactions may help mediate the observed adhesion. Keywords: Adhesion; Dental plaque; Epithelial cells; Saliva; Amphipathic agents.

A Quantitative Immunofluorescence Study of the Association between Streptococcus mutans and Approximal Caries

Abstract: Indirect immunofluorescence staining, using a high-titred polyclonal antiserum specific for Streptococcus mutans , was used to identify and enumerate the proportions of this organism in 320 small samples of approximal plaque from 71 children's premolars. The marked sample sites were correlated to the positions of visible lesions beneath the plaque and the proportions of S. mutans analysed. S. mutans was undetected ( 0.2 per cent of total counts) in 21.6 per cent of approximal plaque samples. The detection of S. mutans was very site-specific; on occasions it could be detected at one approximal site but not at adjacent ones. There was a significant association between the presence of S. mutans and caries lesions on an individual site basis (p< 0.05). Keywords: Caries; Immunofluorescence; S. mutans .

Effect of microcolony formation on the adherence of pseudomonas aeruginosa to human buccal epithelial cells

Abstract: Pseudomonas aeruginosa is thought to initiate respiratory infections in susceptible individuals by adhering to and colonising the respiratory epithelium. We have examined the binding properties of P. aeruginosa strain P1 to human buccal epithelial cell (BEC)s and found that it may bind as a single bacterium or as an alginate encased microcolony. Examination of the binding of P1 to BECs revealed that single bacteria bound to BECs via a single class of adhesinreceptor site interactions, where the apparent association constant of binding (K a ) was 4.6 x 10 -9 ml/CFU and the maximum number of bacteria that could bind per BEC (N) was 223 bacteria/BEC. Cultures that produced microcolonies bound to BECs via two classes of adhesin-receptor site interactions. The first was identical to the class employed by single bacteria and was due to the presence of single bacteria in the culture and did not represent microcolony binding to the BECs. The second class of adhesin-receptor site interactions had a K a of 4.7 x 10 -10 ml/ CFU and N of 3388 bacteria/BEC. This second class of interactions was due to the binding of microcolonies to BECs. These data indicated that single bacteria bound to the BEC with a higher affinity than microcolonies. While the adherence of microcolonies to BECs was not as efficient as single cells, substantially higher numbers of bacteria could attach to an epithelial cell. Keywords: Pseudomonas aeruginosa ; Bacterial colonisation; Bacterial binding; Microcolony.

Bacterial Metabolism in Humans of the Carcinogen IQ to the Direct Acting Mutagen Hydroxy-IQ

Abstract: 7-hydroxy-IQ is the major product of the bacterial metabolism of IQ, a potent dietary carcinogen. Yet, unlike IQ, hydroxy-IQ is directly active in the salmonella/microsomal mutagenicity assay. Two subjects consumed a meal of fried meats containing IQ but no detectable hydroxy-IQ. Hydroxy-IQ was isolated from the subjects’ faeces collected within 30 h following the fried meat meal; it was absent from the subjects’ faeces before and after the meal. This is the first evidence that hydroxy-IQ is formed in humans. Subsequent concern is therefore raised as to its role in the aetiology of colon cancer. From the faeces of one of the subjects a strain was isolated which conformed to the description of a Eubacterium sp. and was capable of producing hydroxy-IQ. Keywords:-2-amino-3-methyl-3 H -imidazo[4,5-ƒ]quinoline;IQ; 2-amino-3,6-3dihydro-3-methyl-7 H -imidazo[4,5-ƒ]-quinoline-7-one; Hydroxy-IQ; Intestinal flora; Carcinogens; Faecal Mutagens; Clostridium ; Eubacterium moniliforme .

Marine Oils in the Modulation of Colonic Flora and pH: Considerations for Colon Cancer

Abstract: BALB/c mice fed high fat diets containing coconut, safflower, or menhaden marine oil were evaluated for faecal flora and faecal pH. Animals fed marine oils showed significantly higher total aerobic Gram-negative rods at 10 9 CFU/gm, compared with those fed saturated or polyunsaturated fat diets with lo6 CFU/gm. Marine oil fed mice exhibited a significantly lower log 10 ratio of anaerobes:aerobes at 1.17, compared to the saturated and polyunsaturated fat fed mice at 3.49 and 2.87 respectively. Differences in flora composition were seen, with increased E. coli (10 9 CFU/gm) for the marine oils group compared to the saturated and polyunsaturated groups, each with lo6 CFU/gm. Faecal samples from animals on marine oils exhibited a lower pH (6.1), compared to samples from animals on saturated or polyunsaturated fat with pH of 7.32 and 7.49 respectively. β-glucuronidase levels were also evaluated and showed no significant differences among the different diets. Keywords: Colonic flora; Marine oils; Dietary lipids; Faecal pH; β-glucuronidase; Colon cancer.