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C W Keevil

Publications and source records attributed to C W Keevil.

At least 55 records · Page 3Linked to original sources
PubMed1991

Development of the BIOLOG substrate utilization system for identification of Legionella spp.

The genus Legionella consists of 51 serogroups comprising 34 species. Biochemical reactions and cell wall fatty acid and quinone analyses may confirm that an isolate is a Legionella sp. and indicate to which species it belongs, but DNA hybridization studies have been necessary for a definitive identification. Recently, the commercially available BIOLOG identification system has offered a standardized, easily reproducible system of substrate metabolism by bacteria resuspended in multiwell plates. A tetrazolium dye acts as an electron acceptor during the oxidation of the wide range of substrates and forms an irreversible, highly colored formazan when reduced. The 95 substrate wells are read rapidly with a conventional plate reader, and the results are downloaded for comparison with a computer data base, allowing quick identification. The BIOLOG system's ability to test more diverse classes of substrates, including amino acids, peptides, carboxylic acids, and carbohydrates, was used in this study to establish a new data base and identify the asaccharolytic Legionella spp. In particular, Legionella pneumophila behaved as a microaerophile, and the fastest, most diverse metabolic activities occurred after the development of a low-oxygen incubation environment. Alternatively, bacteria could be successfully incubated in air when their concentration was double that recommended by the manufacturer. Similar results were obtained by using either Page's amoebal saline or distilled water as the resuspending and incubation medium. Type strains did not cross-identify with any of the strains already in the manufacturer's data base. The results indicate that this modified system has value in being able to identify Legionella isolates to the species level.

Bacterial Typing Techniques↗
PubMed1989

Influence of iron-limited and replete continuous culture on the physiology and virulence of Neisseria gonorrhoeae.

Neisseria gonorrhoeae strains P9-2 (PenS) and KW2 (PenR) were grown in chemostats of nonferrous design at constant growth rate, pH and dissolved oxygen tension. Iron limitation (micromax 0.1 h-1) was imposed by omitting iron salts from the defined medium and titrating increasing concentrations of the non-metabolizable iron chelators ovotransferrin and Desferal, to progressively decrease the growth yield. Metabolic activity during iron limitation was very high, with a qGlc which was 2- or 11-fold greater than during cystine- or glucose-limited growth, respectively. More aspartate and isoleucine were metabolized during cystine-limited growth, while more glutamate, proline and serine were metabolized during glucose- or iron-limited growth. Significant concentrations of alanine or valine were excreted during cystine- or glucose-limited growth, respectively. Iron-limited growth of an initial inoculum of non-piliated, transparent colony-forming (P-O-) gonococci resulted in the selection of 100% piliated bacteria. Initial inocula of P+O- gonococci retained this phenotype for over 100 generations. Iron-limited gonococci were extremely virulent in the guinea-pig subcutaneous chamber model and inocula of even 12 bacteria grew rapidly and persisted. By contrast, cystine-limited (iron-replete) gonococci retained piliation but did not survive in the chambers. Transition from iron-limited to glucose-limited growth resulted in marked loss of piliation but the bacteria remained virulent. Loss of virulence did not correlate with susceptibility to killing by normal human serum, nor with changes in the content or composition of lipooligosaccharide, which contained 2.9, 3.7, 4.3 and 4.8 kDa moieties. Additional proteins were detectable in Sarkosyl-purified outer membranes of iron-limited gonococci but several proteins with molecular masses similar to those described in the literature for iron-restricted gonococci were detectable in cystine- or glucose-limited bacteria.

Bacterial Outer Membrane Proteins↗
PubMed1989

Competition between Neisseria gonorrhoeae and Staphylococcus epidermidis during iron-limited or replete continuous culture.

Neisseria gonorrhoeae strain P9-2 was grown in iron-limited or replete continuous culture at a dilution rate of 0.05 h-1, in the presence and absence of Staphylococcus epidermidis. Gonococci maintained expression of pili (P+) and the transparent colony phenotype in pure culture during transitions of iron- and cystine-limited growth. They competed well with staphylococci during iron-limited co-culture and comprised greater than 95% of the population. Transition to cystine-limited growth allowed the staphylococcus to predominate but the gonococcus did not wash out. Furthermore, the gonococcal opaque colony phenotype (O+), indicating synthesis of outer membrane proteins II, was now expressed. Restoration of iron limitation returned the co-culture to its original composition but with P+O+ gonococci dominating. These results suggest that environments might exist in Man where gonococci can compete successfully with normal indigenous bacteria during infection.

Iron↗
PubMed1987

The relationship between glycosyltransferase production and membrane fatty acid composition of Streptococcus sanguis NCTC 7865 grown in the presence of protonmotive force inhibitors.

The fatty acid composition of Streptococcus sanguis NCTC 7865 was not altered by changing the cation composition (Na+/K+) of the growth medium; glucosyltransferase (GTF; EC 2.4.1.5) also remained constant. In contrast, fructosyltransferase (FTF-S; EC 2.4.1.10) production was reduced by at least 50% in medium with a high Na+ concentration. Growth in the presence of ionophores (gramicidin, nigericin or valinomycin) resulted in an increased proportion of saturated fatty acids, principally octadecanoic acid (C18:0), while the proportion of unsaturated fatty acids, predominantly octadecenoic (C18:1) and hexadecenoic (C16:1) acids, decreased. GTF-S production was reduced in the presence of ionophores whereas FTF-S production was completely abolished. Tween 80 significantly increased both GTF-S production and the proportion of unsaturated fatty acids in the cytoplasmic membrane; FTF-S production was unaltered by Tween 80. The production of GTF-S was inversely proportional to the C18:0:C18:1 fatty acid ratio of the cytoplasmic membrane. It was concluded that FTF-S production is directly influenced by protonmotive force (pmf), whereas GTF-S production is affected more by the physical properties of the cytoplasmic membrane, in particular its fatty acid composition. However, as perturbations in pmf generation can lead to variations in membrane fatty acid composition it can be argued that pmf indirectly influences GTF production by changing the saturated:unsaturated or C18:0:C18:1 fatty acid ratio of the cytoplasmic membrane.

Cations↗
PubMed1987

Characterisation of and polysaccharide production by amoxycillin-resistant streptococci.

Small numbers of bacteria capable of growing on agar supplemented with amoxycillin 40 mg/L were isolated from the saliva of 9 out of 20 adult volunteers in a previous study. All the bacteria were identified as Streptococcus sanguis although no strains produced dextran in conventional tests. However, using a specific assay, all the antibiotic-resistant strains were found to secrete glucosyltransferases (GTF), the enzymes that synthesise these extracellular polysaccharides; the production of GTF-S, the enzyme that synthesizes dextran, was 22-43% less than that of an antibiotic-sensitive control strain. Enzyme production by both antibiotic-resistant and sensitive bacteria was markedly inhibited by dextran primer. The amoxycillin-resistant bacteria were resistant to other penicillins; their resistance to erythromycin was variable but they were uniformly sensitive to cephalothin and clindamycin. As dextran production has been proposed as a key factor in the colonisation of damaged heart valves by bacteria such as S. sanguis, these highly resistant bacteria may not pose a threat to the susceptible individual.

Amoxicillin↗
PubMed1987

Plasmid stability and antibiotic resistance of Neisseria gonorrhoea during glucose-limited continuous culture.

Clinical isolates of Neisseria gonorrhoeae harbouring resistance (R) plasmids of mol. wts 4.4 x 10(6) (Asian) or 3.2 x 10(6) (African) were grown in prolonged glucose-limited continuous culture to determine the segregation efficiency of each type of plasmid and their expression of penicillinase activity in the absence of antibiotic selective pressure. One strain contained the African plasmid and cryptic and conjugative plasmids, which were all retained after 96 generations in the chemostat. By contrast, the other strain lost all plasmids after 100 generations. Both strains showed increased sensitivity to a range of antibiotics, particularly to the penicillins. Loss of penicillinase activity as minimal inhibitory concentration decreased was confirmed for both strains by assaying the enzyme spectrophotometrically. Activity decreased with the number of generations and none was detectable at the time of complete plasmid loss. This decrease was apparently due to individual bacteria ceasing to produce enzyme rather than a gradual decline in production by the whole population. The sensitivities to a broad range of antibiotics also generally increased during glucose-limited growth, but one strain became more resistant to clindamycin and the other to tetracycline.

Anti-Bacterial Agents↗
PubMed1987

Microbial film formation: dental plaque deposition on acrylic tiles using continuous culture techniques.

A chemostat system has been developed to model the attachment of oral bacteria, and the subsequent development of plaque film, to acrylic surfaces immersed in steady state cultures. Plaque was removed from the teeth and gingival margin of volunteers who refrained from oral hygiene for at least 72 h. Samples were pooled and inoculated into a complex growth medium maintained at 37 degrees C. Glucose-limited continuous culture was established at a dilution rate of 0.05/h and at pH 7.0. Microbiological analysis of the culture indicated that a complex community of oral bacteria was established, typical of that found in dental plaque. Acrylic tiles were immersed in the fermenter through a modified fermenter head and incubated therein for up to 21 d. Scanning electron microscopy showed that either side of the tiles contained a rough and a smooth surface and these initially favoured the attachment of fusiform bacteria, particularly on the rough surface. Cocci attached to those surfaces which were not heavily colonized by the fusiforms and eventually grew into and on the colonial sheets of the fusiforms.

Adult↗
PubMed1986

Protonmotive force driven 6-deoxyglucose uptake by the oral pathogen, Streptococcus mutans Ingbritt.

Streptococcus mutans Ingbritt was grown in glucose-excess continuous culture to repress the glucose phosphoenolpyruvate phosphotransferase system (PTS) and allow investigation of the alternative glucose process using the non-PTS substrate, (3H) 6-deoxyglucose. After correcting for non-specific adsorption to inactivated cells, the radiolabelled glucose analogue was found to be concentrated approximately 4.3-fold intracellularly by bacteria incubated in 100 mM Tris-citrate buffer, pH 7.0. Mercaptoethanol or KCl enhanced 6-deoxyglucose uptake, enabling it to be concentrated internally by at least 8-fold, but NaCl was inhibitory to its transport. Initial uptake was antagonised by glucose but not 2-deoxyglucose. Evidence that 6-deoxyglucose transport was driven by protonmotive force (delta p) was obtained by inhibiting its uptake with the protonophores, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazine, gramicidin and nigericin, and the electrical potential difference (delta psi) dissipator, KSCN. The membrane ATPase inhibitor, N,N1-dicyclohexyl carbodiimide, also reduced 6-deoxyglucose uptake as did 100 mM lactate. In combination, these two inhibitors completely abolished 6-deoxyglucose transport. This suggests that the driving force for 6-deoxyglucose uptake is electrogenic, involving both the transmembrane pH gradient (delta pH) and delta psi. ATP hydrolysis, catalysed by the ATPase, and lactate excretion might be important contributors to delta pH.

Deoxy Sugars↗
PubMed1986

Physiology and virulence determinants of Neisseria gonorrhoeae grown in glucose-, oxygen- or cystine-limited continuous culture.

Piliated Neisseria gonorrhoeae forming small, transparent colonies (P+O-) on clear typing agar have been grown in prolonged continuous culture to ascertain how different growth environments might affect gonococcal physiology and the expression of virulence determinants. Virulence of the penicillin-sensitive P9-2 and the penicillin-resistant KW1 strains was assessed by their ability to survive in polypropylene chambers implanted into the flanks of guinea pigs. Initial continuous culture experiments in the defined medium of Manchee et al. (FEMS Microbiology Letters 7, 115-118, 1980) indicated that growth was actually cystine-limited, rather than the anticipated glucose-limited. Surprisingly, cysteine was not completely metabolized and ammonium salts remained in excess. The molar growth yield on glucose (YGlc) was 65 g dry wt mol-1 and 45% of the glucose carbon metabolized was converted to biomass. Gonococci, whilst retaining the P+O- phenotype for over 100 generations of growth, did not survive in the subcutaneous chambers when inoculated at a variety of doses. When the cystine and glucose concentrations were increased and decreased respectively, growth became glucose-limited, the YGlc increased to 108 g mol-1 for strain KW1 and 75% of the metabolized glucose carbon was converted to biomass. After 17 generations of growth, however, only 2% of the gonococci retained the P+O- phenotype and P-O- bacteria predominated. Nevertheless, these bacteria were virulent in the chamber model, as was strain P9-2, which also retained only 2% of the P+O- phenotype during glucose-limited continuous culture. By contrast, the P+O- phenotype was retained during prolonged cystine- or oxygen-limited growth but only the latter was virulent. SDS-PAGE of membrane extracts confirmed that opaque colonies (O+) selected from the glucose-limited cultures contained a heat-modifiable protein (protein II) whereas transparent colony types lacked such proteins. The initial phenotype of virulent gonococci recovered from the subcutaneous chambers was P+O- but opaque variants dominated after several days. A 40 kDa outer-membrane protein was apparently induced during oxygen-limited continuous culture whereas a 44 kDa protein was absent during cystine-limited growth.

Animals↗
PubMed1985

Environmental regulation of carbohydrate metabolism by Streptococcus sanguis NCTC 7865 grown in a chemostat.

Carbohydrate metabolism by the oral bacterium Streptococcus sanguis NCTC 7865 was studied using cells grown in a chemostat at pH 7.0 under glucose or amino acid limitation (glucose excess) over a range of growth rates (D = 0.05 h-1-0.4 h-1). A mixed pattern of fermentation products was always produced although higher concentrations of lactate were formed under amino acid limitation. Analysis of culture filtrates showed that arginine was depleted from the medium under all conditions of growth; a further supplement of 10 mM-arginine was also consumed but did not affect cell yields, suggesting that it was not limiting growth. Except at the slowest growth rate (D = 0.05 h-1) under glucose limitation, the activity of the glucose phosphotransferase (PTS) system was insufficient to account for the glucose consumed during growth, emphasizing the importance of an alternative method of hexose transport in the metabolism of oral streptococci. The PTS for a number of sugars was constitutive in S. sanguis NCTC 7865 and, even though the cells were grown in the presence of glucose, the activity of the sucrose-PTS was highest. The glycolytic activity of cells harvested from the chemostat was affected by the substrate, the pH of the environment, and their original conditions of growth. Glucose-limited cells produced more acid than those grown under conditions of glucose excess; at slow growth rates, in particular, greater activities were obtained with sucrose compared with glucose or fructose. Maximum rates of glycolytic activity were obtained at pH 8.0 (except for cells grown at D = 0.4 h-1 where values were highest at pH 7.0), while slow-growing, amino acid-limited cells could not metabolize at pH 5.0. These results are discussed in terms of their possible significance in the ecology of dental plaque and the possible involvement of these bacteria in the initiation but not the clinical progression of a carious lesion.

Amino Acids↗
PubMed1985

Effect of environmental conditions on the fluoride sensitivity of acid production by S. sanguis NCTC 7865.

Growth and environmental conditions affected the fluoride (F) sensitivity of acid production by Streptococcus sanguis NCTC 7865. Cells grown glucose-limited in a chemostat were generally more sensitive than those harvested from cultures in which there was an excess of glucose (amino acid-limited). There was no consistent relationship between the growth rate of cells and their F sensitivity. Slower-growing cells (mean generation time = 14 hr) were more sensitive than those growing quickly when glucose was the limiting nutrient, whereas the faster growing cells from the glucose-excess culture were most susceptible. The pH of the environment markedly affected the F sensitivity of cells: 2 mM F- was sufficient to abolish acid production by cells incubated at pH 5.0, whereas 24 mM F- did not totally inhibit glycolysis at pH 7.0 or 8.0. Regardless of pH and growth conditions, the cationic composition of the environment had the most pronounced effect on acid production and fluoride sensitivity. Cells washed and re-suspended in KCl were more acidogenic and more sensitive to F than the same cells treated with saline. At pH 7.0 and 8.0, saline-washed cells were comparatively unaffected by F, while glycolysis by the same cells at the same pH but washed in KCl could be inhibited by up to 80%. These results suggested that F inhibition could not be explained merely on the basis of HF uptake at low pH values. Since it has been shown previously that the activity of the energized membrane is maintained by K+ and dissipated in the presence of Na+, it was proposed that proton motive force (pmf) might be involved in the uptake of F-.

Acids↗
PubMed1984

Comparison of polyvinyl chloride membrane electrodes sensitive to alkylphosphonium ions for the determination of the electrical difference (delta psi) of Streptococcus mutans and Lactobacillus casei.

Polyvinyl chloride membrane electrodes sensitive to tetraphenyl phosphonium (TPP+), butyltriphenyl phosphonium ( bTPP +), and methyltriphenyl phosphonium ( mTPP +) ions have been compared for the determination of the electrical potential difference (delta psi) of the oral bacteria, Streptococcus mutans DR0001 /6 and Lactobacillus casei RB1014 . All three types of electrode proved suitable for determining delta psi, although the TPP+-sensitive electrode was particularly susceptible to interference by protonmotive force (delta p) dissipators known to inhibit sugar uptake by the bacteria. The mTPP +-sensitive electrode was the least affected. Similarly, both strains had a high nonspecific binding capacity for TPP+ and bTPP + ions, and this increased for all three ions when the bacteria were heated to 80 degrees C for 1 h to abolish glucose uptake and metabolism. This heat-treatment procedure is therefore not a suitable control for determination of nonspecific binding to cells. However, 1% (v/v) toluene, 20 microM gramicidin, or 10 microM valinomycin effectively depolarized the bacteria without interfering with nonspecific binding. The ionophores were therefore used subsequently for the determination of nonspecific binding of the lipid-soluble cations. The mTPP + ion and corresponding electrode proved the most effective system, and delta psi values of -89 and -107 mV were obtained for S. mutans and L. casei, respectively, harvested from glucose-limited continuous cultures and incubated in 100 mM Hepes-KOH buffer (pH 7.0), containing 1 mM dithiothreitol and 10 mM glucose. Although the delta psi of S. mutans decreased significantly in the presence of Mes-KOH and potassium phosphate buffers at pH 7.0, it increased to -119 mV in Tris-HCl buffer (pH 7.0).(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport, Active↗
PubMed1984

Evidence that glucose and sucrose uptake in oral streptococcal bacteria involves independent phosphotransferase and proton-motive force-mediated mechanisms.

Sugar transport and glycolysis in Streptococcus sanguis NCTC 7865, Streptococcus mitis ATCC 903, Streptococcus salivarius NCTC 8606 and several strains of Streptococcus mutans were investigated by following the rate of acid production by washed bacteria at a constant pH of 7.0. The phosphoenolpyruvate-phosphotransferase system (PTS) was inhibited by low concentrations of chlorhexidine. When this PTS-inhibitory concentration of chlorhexidine was added to cells washed and re-suspended in KCl, glucose uptake and glycolysis continued at a greatly-reduced rate. Chlorhexidine abolished glucose and sucrose uptake and metabolism in bacteria washed and incubated in saline. The Na+-inhibition was reproduced in KCl-washed bacteria using the cyclic peptide ionophores, valinomycin and gramicidin, to dissipate K+ and H+ gradients across the cell membrane. Glucose metabolism by Strep. mutans B13 was more resistant to chlorhexidine than that of Strep. mutans NCTC 10449 or Strep. sanguis but was more sensitive to the ionophores. Valinomycin had a greater inhibitory effect on strain B13 than the other two. That ion gradients are important in the chlorhexidine-resistant glucose-uptake mechanism was confirmed using the classical uncoupling agents, carbonylcyanide-m-chlorophenylhydrazone, 2,4-dinitrophenol and KSCN. Glucose metabolism was inhibited in the presence of both the uncouplers and the PTS-inhibitory concentration of chlorhexidine and significant inhibition was also observed in the absence of the PTS inhibitor. Lactate or the ATPase inhibitor, dicyclohexyl carbodiimide (DCCD), had similar inhibitory effects on the non-PTS uptake system.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1984

Inhibition of the synthesis and secretion of extracellular glucosyl- and fructosyltransferase in Streptococcus sanguis by sodium ions.

The influence of Na+ and K+ on the synthesis and secretion of extracellular glucosyltransferase (GTF; EC 2.4.1.5) and fructosyltransferase (FTF; EC 2.4.1.10) by Streptococcus sanguis NCTC 7865 and Streptococcus sanguis Challis NCTC 7868 has been determined. No FTF and little or no mutansucrase (GTF-I) activities were detectable during growth on glucose or sucrose unless the Na+/K+ ratio of the cultures was kept low. Increasing K+ concentrations stimulated the production of FTF and dextransucrase (GTF-S), but all glycosyltransferase activities decreased in high K+ media when the growth pH was maintained with NaOH instead of KOH, indicating that the Na+/K+ ratio effect was due principally to Na+ inhibition. Significant GTF and FTF activities were detected in a putative GTF- mutant of strain Challis grown in high K+ medium but not in high Na+ medium, suggesting that the mutant might be defective in a regulatory gene.

Culture Media↗
PubMed1984

Regulation of glucose metabolism in oral streptococci through independent pathways of glucose 6-phosphate and glucose 1-phosphate formation.

In vivo rates of glucose uptake and acid production by oral streptococci grown in glucose- or nitrogen-limited continuous culture and batch culture were compared with the glucose phosphorylation activities of harvested, decryptified cells. The strains examined contained significant phosphoenolpyruvate-phosphotransferase system (PTS) activity, measured by a glucose 6-phosphate (G6P) dehydrogenase-linked assay procedure, but this activity was insufficient to account for the in vivo glucose uptake rates. However, ATP was a superior phosphoryl donor to phosphoenolpyruvate, and unlike the PTS, phosphoryl transfer with ATP was insensitive to bacteriostatic concentrations of chlorhexidine, suggesting glucokinase-mediated G6P formation. Again, G6P formation from the PTS and glucokinase reactions was not commensurate with some of the glucose uptake rates observed, implying that other phosphorylation reactions must be occurring. Two novel reactions involving carbamyl phosphate and acetyl phosphate were identified in some of the strains. No G6P formation was detected with these potential phosphoryl donors, but in the presence of phosphoglucomutase, glucose 1-phosphate (G1P) formation was evident, which was insensitive to chlorhexidine. G1P is a precursor of glycogen, and good correlation was obtained between G1P formation activity and endogenous metabolism of washed cells measured either as a rate of acid production at a constant pH 7 or as a decrease in pH with time in the absence of titrant. A "league table" of abilities to synthesize G1P and produce acid from endogenous metabolism was compiled for oral streptococci grown in batch culture. This indicated that Streptococcus mutans Ingbritt and Streptococcus sanguis Challis were unable to form G1P or produce much acid endogenously, whereas increasing activities were obtained with Streptococcus salivarius, Streptococcus sanguis, and Streptococcus mitis. In particular, S. mitis had the highest G1P formation activities and was able to decrease the pH to less than 5 in 15 min by endogenous metabolism alone. The data are consistent with the intracellular accumulation of free glucose driven by proton motive force when PTS activities are low and the subsequent phosphorylation to either G6P for metabolism via glycolysis or G1P for glycogen biosynthesis. The accumulation of acetyl phosphate during glucose-limited growth and the availability of arginine for catabolism to carbamyl phosphate provide an explanation as to why some glucose-limited oral streptococci continue to synthesize glycogen under these conditions, which might prevail in plaque.

Chlorhexidine↗
PubMed1984

Relationship of bioenergetic processes to the pathogenic properties of oral bacteria.

The energized membrane has been shown to affect properties (sugar transport, acid production, intracellular polysaccharide formation, and glycosyltransferase secretion) related to the pathogenicity of oral bacteria. The activity of the energized membrane was susceptible to modulation by environmental conditions likely to be encountered by bacteria in dental plaque.

Acids↗