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Laboratory studies of ureolytic nitrification were carried out to determine whether the ability of ammonia oxidisers to hydrolyse urea could explain their persistence and activity in acid soils. Ammonia oxidising bacteria were isolated from a number of acid soils, using previously described and novel techniques, and isolates tested for their ability to hydrolyse urea. None of the 17 isolated strains were found to be ureolytic, nor were they active below pH 7, indicating the persistence of neutrophilic ammonia oxidisers in acidic soils. The failure to isolate ureolytic and acidophilic strains suggested either their absence in these soils or inadequacies with the isolation procedure. Ten strains of ammonia oxidisers, previously isolated by other workers, were also tested for ureolytic activity and two were found to be ureolytic, Nitrosospira sp. (NPAV) and Nitrosospira sp. The growth of Nitrosospira sp. (NPAV) in liquid batch culture was studied in buffered and unbuffered media revealing that, in the presence of urea, growth and activity could be maintained in media with a pH value of 4-7 whereas growth on ammonium sulphate only occurred at or above pH 7. This suggested that ureolytic strains were capable of growth and activity in acidic conditions if urea was present, providing an explanation for the nitrification in acid soils. The oxidation of urea to nitrite by cultures was incomplete and ammonium accumulated. Growth appeared to inhibited at pH 8 in some media suggesting inhibition of growth by urea in these conditions. The growth and activity of Nitrosospira sp. (NPAV) was studied in continuous flow columns at low pH. Activity could be initiated in continuous flow columns by medium containing urea at pH 4 whereas ammonia was only oxidised at or above pH 6 when medium containing ammonium sulphate was supplied. When effluent nitrite production was constant and a steady state had been established, urea was completely hydrolysed by Nitrosospira sp., causing an increase in the pH, indicating the formation of NH3.

Background The inexorable rise in antimicrobial resistance (AMR) interferes with the goals of health care services around the world, given how critical the antibacterials are in making infections treatable and surgical procedures doable. Nursing homes residents have been identified as a reservoir for AMR, possibly due to the combination of being physically and mentally frail, frequently treated with antibacterials, and frequently moved between nursing home and hospital. Microbiological surveillance is a key countermeasure against further AMR development. Yet, surveillance data is easily biased due to precision problems regarding how the data is collected and evaluated. Methods Beginning in 2008, we launched two programmes (“SHADES” and “MIDIO”) aimed to gathering AMR data in a systematic fashion from elderly nursing home residents and elderly people living in their own place of residence. In doing so, we focused on colonizing strains of the two most important nosocomial infectious agents, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). The bacteria were collected from multiple body sites and analysed with respect to antimicrobial susceptibility and genetic diversity. Results Active surveillance of AMR showed that (i) a S. aureus isolate could be retrieved from 1 in every 2 individuals given a single round of sampling, but aggregating several rounds of sampling, this figure might reach 7 in every 10 individuals, (ii) an E. coli isolate could be retrieved from 4 in every 5 individuals, (iii) the overall prevalence of AMR was favourable when compared to the situation in many other countries, (iv) the genetic diversity of S. aureus was generally high and provided only limited evidence of clonal expansion or contraction, and (v) diabetes mellitus was one of very few patient-level factors to show an association with the degree of genetic diversity in S. aureus. Conclusions The prevalence of colonization with S. aureus and E. coli was somewhat higher than expected, but the degree of AMR was very low. The genetic diversity of S. aureus was generally high. Diabetes mellitus emerged as the only patient-level factor associated with a higher degree of genetic diversity in S. aureus.

This thesis describes the pathway of sulphur metabolism in Paracoccus denitrificans (NCIB 89MJ-) The compounds involved in the sulphur metabolic pathway were determined in growth experiments (Chapter 2) and time-course and pulse-chase experiments, using radioactively labelled sulphate (Chapter 3). P. denitrificans is an assimilatory sulphate-reducing organism with a pathway of sulphate reduction involving in- organic intermediates. P. denitrificans can not utilise cysteine as a sole sulphur; cysteine inhibits amino acid metabolism at low concentrations and respiration at higher concentrations. Sulphate is taken up by P. denitrificans against a concentration gradient (Chapter 4.) . The mechanism of sulphate uptake was investigated using right side out and inside out membrane vesicles prepared from P. denitrificans. The uptake mechanism involves an uncoupler-sensitive transport mechanism driven either by respiration, or by a transmembrane pH gradient (alkaline inside). The active transport of sulphate was shown to be carrier-mediated, by its sensitivity to sulphydryl-group reagents. It is proposed that the sulphate carrier operates by a mechanism of electroneutral proton symport, and is capable of transporting sulphate in either direction across the plasma membrane (Chapter 5). ATP Sulphurylase, the initial enzyme involved in the activation of sulphate, was purified, and its kinetic and regulatory properties investigated (Chapter 6). ATP Sulphurylase activity was repressed by sulphite, sulphide and cysteine, and inhibited by sulphide. Accumulation of APS, the end-product of the ATP sulphurylase-catalysed reaction, could only be detected in the presence of inorganic pyrophosphatase, an enzyme which removed pyrophosphate, another end-product of sulphate activation. Inorganic pyrophosphatase was purified and its substrate specificity, kinetics and regulatory properties examined, in relation to its part in sulphate metabolism (Chapter 7). Inorganic pyrophosphatase is a constitutive enzyme which functions equally well with either Mg²⁺ or Co²⁺ as the cofactor. APS Kinase activity was detected in crude extracts of P. denitrificans. A new assay is described for measuring APS kinase activity (Chapter 8). APS Kinase was purified. Coupled enzyme assays, with purified ATP sulphurylase, inorganic pyrophosphatase and APS kinase, indicated that all three enzymes were necessary for the synthesis, and accumulation of PAPS (Chapter 8). No 3andprime;-nucleotidase or enzyme "A" activity was detected. Serine transacetylase and O-acetyl serine sulphydrylase were purified and the kinetics and regulation of these two enzymes, investigated (Chapter 9). O-Acetyl serine lyase activity was detected in crude extracts of P. denitrificans, representing the first report of this enzyme in bacteria (Chapter 9). β-Cystathionase was purified and its kinetic and regulatory properties investigated; the unidirectionality of the cysteine to methionine pathway was confirmed (Chapter 10). Cysteinyl- and methionyl-tRNA synthetases were purified and the kinetics and regulation of these enzymes studied (Chapters 11 and 12 respectively). Both these enzymes possess different substrate specificities to the aminoacyl-tRNA synthetases from other organisms. Both enzymes appear to be constitutive. During this investigation of the sulphur metabolism of P. denitrificans, the substrate specificity of the different enzymes, to the selenium analogues of the respective sulphur-containing substrates, were investigated. Selenate competitively inhibits sulphate uptake and ATP sulphurylase, with respect to sulphate, but no APSe or PAPSe synthesis could be detected in the coupled enzyme assays (Chapters 7 and 8). Purified O-acetyl serine sulphydrylase catalysed the synthesis of selenocysteine from selenide and O-acetyl serine (Chapter 9). Both selenocysteine and selenomethionine are activated by the respective aminoacyl-tRNA synthetase (Chapters 11 and 12, respectively).

In the work presented here, it is claimed that bacteria have been isolated which are capable of growth at high temperatures utilizing molecular nitrogen as their sole nitrogen source. Soil bacteria were isolated which grew at 55 C in nitrogen-free media. They were found to be obligatory thermophiles in nitrogen-free media and facultative thermophiles in media containing organically bound nitrogen.

It is the purpose of this investigation to modify existing methods in an attempt to provide a rapid identification of members of the genus Streptomyces.

The purpose of this study was to search for the substrates utilized by Azotobacter vinelandii in dialysed soil media. Also, we sought to determine the relationship between these substrates and the growth and morphological variations of A. vinelandii. p-Hydroxybenzoic acid was shown to be used as the carbon and energy source by A. vinelandii in dialysed soil medium. The amount of this compound in the soil dialysed soil medium ranged from 14 to 21 micrograms per gram of soil. In a dialysed soil medium, p-hydroxybenzoic acid induced A. vinelandii to form minute bodies, similar to the filtrable forms reported by Gonzalez and Vela, although no growth of minute bodies was detected.

A detailed kinetic analysis was made of growth of Nitrosomonas europaea and Nitrobacter sp. in chemostat culture. Steady states were established at a number of dilution rates as measured by substrate concentration and cell number. Biomass concentration was also estimated at each dilution rate and these data were used to evaluate the kinetic growth constants; maximum specific growth rate (m), saturation constant (Ks), true growth yield (Yg) and maintenance energy coefficient (me). The transient response to dilution rate changes was monitored and step increases in dilution rate always resulted in substrate overshoots which were mirrored by cell number undershoots. Step reductions in dilution rate resulted in monotonic changes in substrate concentration to lower steady state levels. Nitrobacter exhibited different growth characteristics when cultured continuously in an airlift column fermenter. This was considered to result from biomass settling within the column, resulting from inadequate mixing. Kinetic growth constants for culture of Nitrobacter in the airlift column fermenter were determined. The optimum pH for nitrite oxidation in batch culture was pH 7.5 with no growth at pH values less than 6.0. Nitrite steady states were established in continuous culture at pH 8.0, 6.0 and 5.5, with washout occurring at pH 5.0 and transient nitrite undershoots were observed following pH changes imposed. Surface growth of Nitrobacter was investigated in batch and continuous culture. Attachment and growth on glass and anion exchange resin surfaces resulted in the development of attached microcolonies. Biofilm development on anion exchange resin surfaces was facilitated by slime production which may assist in the irreversible attachment of cells. The specific rate of nitrite oxidation of cells attached to glass surfaces in batch culture was 20-25% greater than that of freely suspended cells. The enhanced activity of attached cells was independent of pH and pH-activity curves of free and attached cells were similar. Similarly cells attached to anion exchange resins in continuous culture exhibited increased oxidation rates per cell, compared to free cells cultured in the same system. Attachment to surfaces significantly lowered the minimum pH for nitrite oxidation and increased protection against low pH, Reduction in pH to 3.5 prevented nitrite oxidation, however cells remained viable and a steady state was subsequently established at pH 4.5.

El objetivo del estudio fue determinar la actividad antibacteriana del extracto de hoja de coca (Erythroxylum coca), sobre Bacilos Negro Pigmentantes (BNP), dichas bacterias son bacilos anaerobios Gram negativos, y están fuertemente relacionadas con la progresión de la Periodontitis; para lo cual se empleó dos pruebas; la primera, el Test de Difusión en Agar y la segunda, la prueba de dilución en medio líquido. Las cepas de BNP fueron aisladas desde muestras tomadas de bolsas periodontales (mayores a 4 mm) de pacientes con enfermedad periodontal atendidos en la Facultad de Odontología de la UNMSM. El extracto de hoja de coca fue obtenido de la Empresa Nacional de la Coca (ENACO). Los resultados de la primera prueba indicaron sensibilidad nula (-) para la mayor parte de las concentraciones evaluadas, y sensibilidad límite (sensibilidad: +) para las concentración de 12,5 % y 100 %. Los resultados del segundo estudio determinaron una concentración mínima del extracto capaz de inhibir el crecimiento de dicha bacteria, 100 % (CMI), y a las concentraciones de 12,5 % y 6,25 % se observa una repotenciación del efecto antibacteriano del extracto. Por lo tanto se concluye, que el extracto de hoja de coca, sí presenta una actividad antibacteriana frente a BNP, a las concentraciones de 100% y 12, 5%. PALABRAS CLAVE: Extracto, coca, antibacteriano, bacterias anaerobias. / --- The aim of this study was to determine the antibacterial activity of the extract of coca leaves (Erythroxylum coca), on Black Pigmented Bacteria (BNP). Those periodontopathogenic bacterias are Gram negative and strict anaerobic, also they are strongly related with the progression of periodontitis. To this purpose, the study used two different kinds of tests; the first, Agar Diffusion Test; and the second, Dilution Test in Liquid Medium. BNP strains were isolated from samples taken from periodontal pockets (greater than 4 mm) of patients with periodontal disease treated at the Faculty of Dentistry of San Marcos University. The coca leaf extract was obtained from the National Coca Company (ENACO). The results of the first study indicated that the extract of Erythroxylum coca has zero sensitivity (-) for most of the tested concentrations, and sensitivity limit (sensitivity: +) to the concentration of 12,5 % and 100 %. The results of the second study determined a minimum concentration of extract able to inhibit the growth of said bacterium; this value was 100% (CMI) Also, concentrations of 12,5 % and 6,25 %, both indicated repowering of the antibacterial effect of the extract. Therefore it is concluded that, the extract of coca leaf has an antibacterial activity against BNP, in concentrations of 100% and 12, 5%. KEY WORDS: Extract, coca, antibacterial, anaerobic bacteria.