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Biomedical subjects

R J Miles

Publications and source records attributed to R J Miles.

11 recordsLinked to original sources

The detection of lipase activity in bacteria using novel chromogenic substrates.

The propionate (Pro), decanoate (Dec) and laurate (Lau) esters of 5-(4-hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothiazoline++ +-3-acetic acid were assessed as substrates for lipase and esterase. On hydrolysis these substrates yield an intensely red coloured phenol which could be assayed at 505 nm. The Pro ester was an effective substrate for porcine esterase and was hydrolysed at a rate 20 times greater than the Lau and Dec esters. Conversely, Pseudomonas lipase had a high activity towards the Lau and Dec esters, especially in the presence of bovine serum albumin, but little activity towards the Pro ester. The Dec and Lau were used to detect lipolytic activity in Pseudomonas strains associated with milk spoilage. For this purpose, the substrates were absorbed onto filter paper disks, which were placed over bacterial colonies growing on agar plates; activity was indicated by bright red colouration of discs within 2 h. Escherichia coli colonies hydrolysed the Pro but not the Lau or Dec esters.

Benzoates

Catabolism in mollicutes.

The small genome size of mollicutes, and particularly mycoplasmas and ureaplasmas, precludes their possession of the extensive range of metabolic activities present in most other bacterial groups. Demonstrated catabolic activities appear primarily to be associated with energy generation, rather than the provision of substrates for synthetic pathways, and anabolism is largely dependent upon extracellular sources of amino acids, nucleic acid precursors and lipids. However, the pathways of energy generation in mollicutes are diverse and specialized, and may in vivo be dependent upon the presence of a single amino acid (arginine) or urea. Even in those species that utilize carbohydrates the range of substrates is restricted, and while Ac. laidlawii has both EMP and PP pathways and is able to oxidize pyruvate to acetate plus CO2, many mycoplasmas possess only a part of these activities. Such specialization and the infrequent demonstration of inducible enzyme activity in mollicutes implies adaptation to specific habitats in host species, and suggests that differences in the catabolic activities of mollicute strains may be significant in terms of their ecology and pathogenicity. The demonstrated energy-generating pathways of mollicutes produce low ATP yields. Thus, mollicute growth will generate relatively large quantities of metabolic end-products and may deplete host tissues of substrates. Arginine depletion may be of particular importance in pathogenesis and the close physical association between mollicutes and host cells will enhance the potential significance of NH4+ production from the hydrolysis of arginine and urea, and of H2O2 and superoxide formation during carbohydrate metabolism. In addition, lipid and protein catabolism may be associated with virulence where extracellular or membrane-bound enzyme activities exist. Membrane-bound DNAase and RNAase activities have also been demonstrated in mycoplasmas and Ac. laidlawii (Pollack et al., 1965) and U. urealyticum (Romano & La Licata, 1978). Many aspects of mollicute catabolism, including energy conservation in some groups, is poorly understood. Also, while substantial catabolic diversity has been demonstrated within mollicutes and new species are continually being isolated, metabolism has been studied in relatively few species, and even in these only single strains or small groups of strains have been used. In this review, therefore, an attempt to avoid generalizations concerning mollicute behaviour has been made. The lack of much basic knowledge concerning mollicute metabolism has also necessitated the widespread use of 'may be' and other equally vague terms.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbohydrate Metabolism

Refinement of the transpalatine exposure of the nasopharynx.

The intrasulcular incision provides excellent transpalatine exposure of the nasopharynx with a viable palatal flap. There is less chance for naso-oral fistula formation since the resulting suture line is always over underlying bone. This safely allows extensive removal of the hard palate. Additionally, the continuous sling suture allows excellent flap reapproximation using the teeth as anchors for the replaced palatal flap. Patients tolerate oral feedings within 24 hours of their operations and require minimal postoperative analgesics. The extended intrasulcular incision offers many advantages over other methods for transpalatine exposures to the nasopharynx.

Gingivectomy

Oxygen uptake and H2O2 production by fermentative Mycoplasma spp.

Oxygen uptake and H2O2 accumulation during the metabolism of glucose and glycerol by whole cells, and of L-alpha-glycerophosphate (GP) and NADH by cells lysed with Triton, was determined for the type strains of six fermentative Mycoplasma species. Oxidation of glucose and of NADH by M. mycoides, M. pneumoniae and M. putrefaciens was accompanied by the accumulation of relatively small quantities of H2O2 (less than 0.05 mol/mol O2), though larger quantities (0.17-0.24 mol/mol O2) were produced by M. dispar. M. fermentans and M. canis were distinguished from the other strains used in that O2 uptake in the presence of glucose could not be demonstrated. However, metabolism of glucose was indicated by a reduction in the pH of the suspending medium and lysed cells oxidised NADH with the production of approximately 1.0 mol H2O2/mol O2 taken up. Glycerol was oxidised by all the strains studied except M. fermentans, and large quantities of H2O2 (0.48-1.07 mol/mol O2) accumulated. Cells of the glycerol-oxidising strains, lysed with Triton, oxidised GP with the production of approximately 1.0 mol H2O2/mol O2 utilised, which indicated the presence of a GP oxidase. The importance of H2O2 production as a factor in the pathogenicity of some mycoplasmas might depend upon the availability of glycerol in vivo.

Glucose

Expression of carbonic anhydrase in neisseriae and other heterotrophic bacteria.

A diverse range of heterotrophic bacteria was screened for the presence of carbonic anhydrase (CA) activity, sensitivity to inhibition of growth by acetazolamide (CA inhibitor), and the presence of protein binding monospecific antibody prepared against purified Neisseria sicca CA. CA activity was demonstrated only in strains of N. sicca and N. gonorrhoeae. However, all Neisseria strains, including various isolates of N. meningitidis and N. lactamica, were sensitive to acetazolamide, when grown in air, and showed serological cross-reaction with N. sicca CA. Strains of other genera were resistant to acetazolamide. A number of strains including members of the genera Pseudomonas, Staphylococcus, Streptococcus, Serratia and Proteus also strongly expressed a gene product(s) immunologically related to CA. The presence of CA cross-reacting proteins, which lack hydrase activity, is discussed in relation to the function of the various mammalian CA isoenzymes.

Acetazolamide

Antibiotic sensitivity testing by flow microcalorimetry.

The proposed flow microcalorimetric method for the diagnosis of bacteriuria has been extended to include antibiotic sensitivity testing. Sensitive organisms rapidly (4-8 min) show thermal responses to the added antibiotics over the normal range of concentrations (1 x, 2x, MIC value).

Anti-Bacterial Agents

The application of cryobiology to the microbiological assay of nystatin.

Improvements in the reproducibility of nystatin agar diffusion assays have been achieved by the use of liquid nitrogen stored inocula and deep frozen standard stock solutions. The overall percentage variability of the assay has been reduced from over 5% with daily prepared standards and inocula to around 1% with a frozen inocula and to 0.6% with a combination of frozen inocula and standards. The implications of these improvements in the standardization of nystatin assays, and microbiological assays generally are discussed.

Biological Assay

A comparative study of the microbiological assays currently available for nystatin raw material.

The classical agar diffusion and turbidimetric methods of assay for nystatin are compared with the more recently documented assays for this antibiotic which depend upon physicochemical measurement of the response of micro-organisms. Liquid nitrogen stored inocula were used throughout. It is concluded that the newer methods of assay are as reproducible and reliable as the agar diffusion and turbidimetric methods and that they are generally more sensitive. The choice between the assay methods compared can thus be based on speed, cost and sample through-put.

Biological Assay

In vitro studies of amphotericin B in combination with the imidazole antifungal compounds clotrimazole and miconazole.

The clinically important polyene antibiotic amphotericin B, in combination with two antifungal imidazole compounds, clotrimazole and miconazole, was studied in vitro. With use of results of cytoplasmic leakage, metabolic heat output, and minimal inhibitory concentration studies, a definite antagonistic response was demonstrated. It is suggested that, if combined antifungal drug therapy is clinically indicated, the drug combination be tested against the isolate by the simple technique of measuring cytoplasmic leakage or by the more elaborate method of flow microcalorimetry.

Adsorption