Roger J. Miles

Trisodium phosphate increases sensitivity of gram-negative bacteria to lysozyme and nisin.

Cell suspensions of Campylobacter jejuni, Escherichia coli, Pseudomonas fluorescens, and Salmonella enteritidis exposed to sublethal concentrations (0.5 to 5 mM) of trisodium phosphate (TSP) for 10 min showed greatly increased susceptibility to lysozyme (10 micrograms ml-1) and/or nisin (1 microM). Under optimal conditions at 37 degrees C, reductions in viable count after 30 min were up to six log cycles. At 4 degrees C, C. jejuni showed greater resistance than at 37 degrees C, and maximal cell kills (95%) were reduced by more than two log cycles. Cells dried on the surface of chicken skin were more resistant than suspended cells to TSP-lysozyme and TSP-nisin treatments; nevertheless, at 37 degrees C, kills varied from approximately 95% for S. enteritidis cells with nisin (30 microM) or lysozyme (100 micrograms ml-1) to > 99.9% for C. jejuni and E. coli cells with nisin. Under the experimental conditions used, nisin also reduced viable counts of skin-attached Staphylococcus aureus by > 99.9%. The results suggest that the high TSP concentrations (approximately 10% wt/vol, 0.25 M) needed for successful decontamination of gram-negative bacteria, on the surface of poultry and other foodstuffs, may be substantially reduced by following TSP treatment with exposure to low lysozyme or nisin concentrations.

Biochemical diversity within the "Mycoplasma mycoides" cluster.

The metabolism of 51 strains within the ‘Mycoplasma mycoides cluster’ was investigated by measuring oxygen uptake following the addition of organic substrates to washed cell suspensions. There were extensive differences between strains in the range of substrates utilized, the relative rates of oxidation and the observed saturation constants for substrates, which ranged from a few μM to several mM. M. mycoides subsp. capri and M. mycoides subsp. mycoides LC (large colony) strains were diverse and could not be distinguished by substrate utilization patterns. However, there were consistent differences in the patterns of substrate utilization between other groups of the M. mycoides cluster, suggesting that these patterns may be useful in identification. In particular, SC (small colony) strains of M. mycoides subsp. mycoides were distinguished by their inability to oxidize maltose, trehalose and (at low concentrations) mannose and glucosamine. Surprisingly, the type strain, M. F38, of Mycoplasma capricolum subsp. capripneumoniae and two further isolates differed from all other strains in that they did not oxidize glucose or other sugars. They did, however, oxidize pyruvate, lactate and 2-oxobutyrate at high rates. The marked metabolic differences between these strains and M. capricolum subsp. capricolum strains is in contrast to the genetic evidence that was used to support the designation of the M. F38 group as a subspecies of M. capricolum.

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.

Oxidation of glycerol differentiates African from European isolates of Mycoplasma mycoides subspecies mycoides SC (small colony)

CONTAGIOUS bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subspecies mycoides sc (small colony), is the most serious cattle disease in Africa causing greater losses than rinderpest. In Tanzania over 14,000 cattle died of CBPP in the first half of 1995, while outbreaks in Botswana in the same year resulted in up to 60 per cent mortality in many herds (Egwu and others 1996). In contrast, CBPP in Europe is far more insidious, the disease being largely chronic with cattle showing few clinical signs and little mortality (Nicholas and others 1996). There are few meaningful data enabling comparison of virulence among M mycoides subspecies mycoides Sc strains, chiefly because virulence can only be measured accurately by expensive animal infections. Nevertheless, experimental infections have shown virulence differences among African isolates (Provost and others 1987). Recently, analysis of copy number and chromosomal location of a DNA insertion sequence, iS 1296, has provided evidence for two clonal lineages among sc strains. Interestingly; one lineage comprises European isolates and the other isolates from Africa and Australia (Cheng and others 1995). In an extensive biochemical study of 37 M mycoides strains, Abu-Groun and others (1994) showed that sc strains differed consistently from M mycoides subspecies mycoides LC (large colony) and M mycoides subspecies capri strains in the pattern of sugars metabolised. However, all strains oxidised glucose and glycerol. The five sc strains included in this previous study were of African and Australian origin and biochemically similar. In contrast, it is reported here that isolates from recent European outbreaks of CBPP lack glycerol-oxidising ability. In the present study, rates of glucose and glycerol oxidation

The pattern and kinetics of substrate metabolism of Campylobacter jejuni and Campylobacter coli.

Aims:  The main aim was to investigate the patterns and kinetics of substrate oxidation by Campylobacter jejuni and C. coli.

Diversity of energy-yielding substrates and metabolism in avian mycoplasmas

The metabolism of organic substrates and production of H2O2, a potential pathogenicity factor, were studied in the type strains of fourteen avian Mycoplasma species, and in low-passage isolates of M. gallinarum, M. gallisepticum, M. iners and M. pullorum. Substrates were added to cell suspensions in Ringer or saline solution and oxygen uptake and/or change in pH monitored. The fermentative species could be sub-divided according to whether O2 uptake did (M. anatis, M. columborale, M. gallisepticum, M. imitans and M. iowae) or did not (M. gallinaceum, M. gallopavonis and M. pullorum) accompany glucose metabolism and the five non-fermentative, arginine-hydrolysing strains according to whether organic acids (lactate, 2-oxobutyrate, pyruvate) were (M. columbinasale, M. columbinum and M. gallinarum) or were not (M. iners and M. meleagridis) oxidized, Lysed cells of strains which consumed O2 during glucose or organic acid metabolism had relatively high NADH oxidase activity (170-950 nmol min-1 mg cell protein-1) and produced 0.02-0.36 mol H2O2 per mol O2 consumed during NADH oxidation. In contrast, strains which did not oxidize organic acids or consume O2 during glucose or organic acid metabolism possessed low NADH oxidase activity (< or = 20 nmol min-1 mg cell protein-1). All arginine-hydrolysing species showed a high affinity (Km value 1-3 microM) towards arginine. The fermentative species similarly showed a high affinity (Km value 2-5 microM) towards glucose, but used only a small number of additional sugars at detectable rates. All M. pullorum strains metabolized sucrose (Km < or = 3 microM). The type-strains of M. gallisepticum and M. imitans were biochemically similar and had high affinities for fructose and mannose. A number of low-passage avain isolates, but none of the type strains, metabolized glycerol and, in lysed cells, oxidized L-alpha-glycerophosphate (GP) with the production of 1 mol H2O2 per mol GP.

Distribution of Bacteria on Hands and the Effectiveness of Brief and Thorough Decontamination Procedures Using Non-medicated Soap

Our perception of the role of hand washing in the clinical situation is based on experimental studies in which test-bacteria are usually inoculated onto the skin surface and removed using hand washing preparations containing antiseptics. In this study, we have investigated the distribution of bacteria on the hands of volunteers and the effectiveness of long (3 minute) and brief (10 second) washes in removing both naturally-occurring and artificially-inoculated bacteria (Micrococcus sp.), using only soap and water. There was a tenfold reduction in median counts of artificially inoculated bacteria following both long and brief washes. However, less than 50% of naturally-occurring bacteria were removed and, for hands previously disinfected by immersion in 70% ethanol, the washing procedure increased bacterial counts. In both unwashed hands, and hands washed following a strict protocol, the mean variation in counts of naturally-occurring bacteria at different sites (wrists, dorsal surface, palmar surface, fingertips and interdigital spaces) was only two-fold. The efficiency of recovery of naturally-occurring organisms was estimated by repeated swabbing, to be more than 60%. The data question the value of typical hand wash procedures recommended by many authorities for use in clinical situations and of the perfunctory hand washes frequently adopted by nursing staff in busy wards. Experimental evidence is required to justify procedures and to identify the precise circumstances in which they are of value.

An in situ method for determining bacterial survival on food preparation surfaces using a redox dye

A simple method is described for the direct enumeration of viable bacteria dried on test surfaces. Inoculated surfaces were overlayed with agar and after incubation nitroblue tetrazolium solution (pale yellow) was used to stain colonies (purple) at the agar‐test surface interface. Stained colonies could be readily detected and counted even against the opaque background of ceramic tile or stainless steel or when present within opaque films of milk or serum. Recovery of bacteria by this method was approximately fivefold greater than using a conventional swabbing procedure. The method was used to demonstrate the marked effect of the composition of the suspension fluid, in which bacteria were dried, and the length of surface exposure upon bacterial survival.

A rapid and sensitive enzyme linked immunofilter assay (ELIFA) for whole bacterial cells

An improved method is described for the detection of Escherichia coli by an enzyme linked immunofilter assay (ELIFA) using nitrocellulose membrane sandwiched between two 96-well plates. The incorporation of a pumping system permits a continuous flow of reagents and/or wash fluids through the membrane and provides an assay procedure capable of detecting 10(3) bacteria per well within 40 min. Quantitative bacterial detection was based on precipitated chromogen determined by scanning densitometry. The procedure represents a significant improvement in assay time and/or sensitivity over previously described ELIFA and ELISA methods for whole bacterial cells.

Survival and nodulating ability of indigenous and inoculated Rhizobium leguminosarum biovar trifolii in sterilized and unsterilized soil treated with sewage sludge.

Rhizobium leguminosarum biovar trifolii was detected in soil from 41 of 47 plots, within nine sewage sludge-treated sites with different soil characteristics and heavy metal contents. However, although population size varied widely, there was no consistent correlation with soil heavy metal concentration. Indigenous populations in 20 plots within four selected sites retained their ability to induce effective nodule formation after incubation of soil in the dark for 165 days. In sterilized (γ-irradiated) soil, Rhizobium survival varied from 0.01% to 95% depending on the soil sample and strain used. Metal-resistant strains with non-mucoid colonies survived less well than mucoid metal-sensitive strains.