Fatty acids and TxA(2) generation, in the absence of platelet-COX-1 activity.

BACKGROUND AND AIMS: Omega-3 fatty acids suppress Thromboxane A(2) (TxA(2)) generation via mechanisms independent to that of aspirin therapy. We sought to evaluate whether baseline omega-3 fatty acid levels influence arachidonic acid proven platelet-cyclooxygenase-1 (COX-1) independent TxA(2) generation (TxA(2) generation despite adequate aspirin use). METHODS AND RESULTS: Subjects with acute myocardial infarction, stable CVD or at high risk for CVD, on adequate aspirin therapy were included in this study. Adequate aspirin action was defined as complete inhibition of platelet-COX-1 activity as assessed by <10% change in light transmission aggregometry to ≥1 mmol/L arachidonic acid. TxA(2) production was measured via liquid chromatography-tandem mass spectrometry for the stable TxA(2) metabolite 11-dehydro-thromboxane B2 (UTxB2) in urine. The relationship between baseline fatty acids, demographics and UTxB(2) were evaluated. Baseline omega-3 fatty acid levels were not associated with UTxB(2) concentration. However, smoking was associated with UTxB(2) in this study. CONCLUSION: Baseline omega-3 fatty acid levels do not influence TxA(2) generation in patients with or at high risk for CVD receiving adequate aspirin therapy. The association of smoking and TxA(2) generation, in the absence of platelet COX-1 activity, among aspirin treated patients warrants further study.

Biochemical and genetic variation in Mycoplasma fermentans strains from cell line, human and animal sources.

AIMS: To investigate the inter-strain variation in (i) substrate utilization and (ii) the restriction fragment length polymorphism (RFLP) pattern based on the distribution of an insertion element (IS1550) in Mycoplasma fermentans strains, and to establish any correlation between subgroups within the species and their source or habitat. METHODS AND RESULTS: Using a sensitive dynamic pH method, the pattern and kinetics of substrate utilization by a panel of 17 M. fermentans strains from various sources was determined. This study correlated the biochemical characteristics of these strains with RFLP patterns based on the distribution of an insertion sequence (IS1550) with the sources of the strains. The test isolates were divided into four major groups according to the pattern of substrates metabolized. Interestingly, two strains isolated from cell lines in RFLP cluster I failed to utilize arginine. Ovine strains showed distinct substrate utilization patterns and produced RFLP patterns not previously encountered. CONCLUSIONS: All strains utilized glucose, but the ability to utilize arginine, fructose and N-acetyl glucosamine varied. There was also some correlation evident between the metabolic data and the RFLP clusters. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has provided a better understanding of the biochemical and genetic diversity of M. fermentans strains from various sources.

Tetrazolium reduction methods for assessment of substrate oxidation and strain differentiation among mycoplasmas, with particular reference to Mycoplasma bovigenitalium and some members of the Mycoplasma mycoides cluster.

AIMS: To apply a rapid nitroblue tetrazolium (NBT) reduction assay of substrate metabolism by mycoplasmas that would help to differentiate Mycoplasmas. METHODS AND RESULTS: Growth, substrate preferences and tetrazolium reduction were assessed for 18 strains of Mycoplasma bovigenitalium and Mycoplasma ovine serogroup 11. NBT reduction was detectable in 1 h with 10(8) CFU ml(-1). Use of alpha-ketobutyrate, lactate and pyruvate to support growth and NBT reduction were correlated: pyruvate was preferred and lactate was used by only four of the 18 strains. Selected members of the Mycoplasma mycoides cluster were also assessed and monotetrazoles tested as alternatives to NBT. The NBT method was applied to a further 19 species. CONCLUSIONS: This simple and reproducible method requires only small amounts of cells, enabling routine assessment of substrate use within 1 h, and the rapid assignment of numerous mycoplasmas to one of six physiological groups. The four physiological groups of M. bovigenitalium and Mycoplasma serogroup 11 strains were indistinguishable from each other, which supports the view that these belong to the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: Strain-specific substrate-utilization patterns by mycoplasmas can be obtained rapidly and reliably. The method has potential as a large-scale semi-automated procedure to monitor numerous strains and substrates simultaneously.

Isolation and immunological detection of Mycoplasma ovipneumoniae in sheep with atypical pneumonia, and lack of a role for Mycoplasma arginini.

Mycoplasma ovipneumoniae NCTC 10151(T) and four new isolates from UK sheep flocks were compared. Only glucose and pyruvate were used as energy sources by the five strains: glucose was the best energy source for the type strain, pyruvate supported better growth of the new strains. Whole cell protein patterns and antigenic profiles showed high similarity between all five strains. The new isolates fell into two groups in ELISA tests. Serum samples from 30 pneumonic sheep were assessed for M. ovipneumoniae infection and Mycoplasma arginini co-infection. Fourteen (out of 30) serum samples were positive for M. ovipneumoniae both by ELISA and immunoblotting. Twelve antigenic proteins of M. ovipneumoniae were detected in infected serum samples: the antigen patterns were unique, with between one and at least seven occurring in any one sample. All serum samples were designated as negative for M. arginini antibodies by both ELISA and immunoblotting.

Cholesterol protects Acholeplasma laidlawii against oxidative damage caused by hydrogen peroxide.

The aim of this study was to determine whether cholesterol, added to the cell growth medium or to cell suspension buffer, could protect Acholeplasma laidlawii cells against the toxic effects of hydrogen peroxide (H(2)O(2)). Variable concentrations of cholesterol (0.05-1.0 mg/ml) were added to the A. laidlawii suspension buffer and to the growth medium. Cells were then washed carefully and incubated with 0.001% (v/v) H(2)O(2) at 37 degrees C for 30 min and the viability was determined. The results indicated that cells were more viable in the presence of cholesterol than were cells grown in the absence of cholesterol. In addition, the oxygen uptake rate resulting from the oxidation of 5.5 mmol/L glucose was 2-fold and 4-fold higher for cells grown in medium supplemented with 0.05 and 0.50 mg/ml cholesterol, respectively, compared to cells grown in a medium with no added cholesterol. These findings indicate that cholesterol might play a role in protecting Mollicutes against the oxidative damage caused by H(2)O(2).

Hydrogen peroxide production by Mycoplasma bovis and Mycoplasma agalactiae and effect of in vitro passage on a Mycoplasma bovis strain producing high levels of H2O2.

Hydrogen peroxide (H2O2) production and oxygen uptake during the oxidation of NADH and L-alpha-glycerophosphate (GP) by lysed cells was determined for the type and field strains of Mycoplasma bovis and M. agalactiae. NADH oxidation by all the strains showed variable production of H2O2 ranging from 0 to 1.21 mol/mol O2 taken up. All strains were unable to oxidize GP, showing absence of GP oxidase activity. Some strains were identified that produced relatively high levels of H2O2 (> 1.0 mol/ mol O2 taken up). In vitro passage of M. bovis strain 119B96 showed reduced H2O2 production: 0.52, 0.16, and 0.07 mol/mol O2 taken up after the 50th, 100th and 200th passages, respectively. SDS-PAGE analysis showed the loss of a protein band of 32 kDa after 50 passages. These preliminary studies show that not only does H2O2 production by potentially pathogenic Mycoplasma spp. vary in the field but also that similar alterations can be induced by passage in culture. In the latter case, at least in one M. bovis strain, this alteration has been shown by SDS-PAGE to be associated with a loss of specific protein production. Further study of these phenomena is essential background for the production of more efficient vaccines for mycoplasmas.

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. METHODS AND RESULTS: Substrate oxidation profiles by 100 strains were determined using oxygen electrode system. All the isolates tested oxidized formate, l-lactate, cysteine, glutamine and serine with high oxidation rates and high affinity but varied in their ability to oxidize citric acid cycle intermediates, aspartic acid and serine. CONCLUSIONS: Based on the oxidation ability of alpha-ketoglutarate, succinate, fumarate and aspartic acid, Campylobacter strains tested were divided into three distinct metabolic categories. The first group was able to metabolize alpha-ketoglutarate, succinate, fumarate and aspartic acid; the second group was unable to oxidize alpha-ketoglutarate; and the third group was unable to oxidize, succinate, fumarate, and aspartic acid. Furthermore, serine oxidation rate enabled the differentiation of C. jejuni and C. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Overall, the results highlights the extensive metabolic diversity between and within Campylobacter species. In addition, the kinetic data of oxidized substrates obtained may improve the isolation procedures of the organism.

Alternative to fluorescence assays to monitor fusion between Acholeplasma laidlawii cells and liposomes.

AIMS: To develop a new technique as an alternative to the fluorescence assays and electron microscopy for the purpose of monitoring the cell-liposome fusion. METHODS AND RESULTS: Acholeplasma laidlawii whole cells did not oxidize Glucose-6-phosphate (G6P) or Fructose-1,6 diphosphate (F1,6DP) as free (unentrapped) substrates, at concentrations 47 and >270 mM, respectively. Lysed A. laidlawii cells oxidized G6P and F1,6DP at lower concentration of 0.8 and 15 mM, respectively. When these substrates were entrapped inside liposomes, at a final concentration of 1.5 mM, and interacted with A. laidlawii whole cells, in an oxygen electrode chamber, an increase in oxygen uptake was evident. This interaction does not have any effect on cell viability. SIGNIFICANCE AND IMPACT OF THE STUDY: The experimental system described here is advantageous over classical fluorescence assays in determining the fate of liposome-entrapped material and raises the possibility of studying the kinetics of metabolic substrates, which are normally excluded from the cell by the cell membrane.

Investigations of outbreaks of contagious caprine pleuropneumonia in Eritrea.

Mycoplasmas were isolated from freeze-dried lung samples from goats from the western lowlands of Eritea suspected of being affected by contagious caprine pleuropneumonia. The goats belonged to two herds in which mortality and morbidity rates were high. Mycoplasma capricolum subsp. capripneumoniae was identified in most samples by the polymerase chain reaction and by conventional serological tests. The latex agglutination test detected more positive serum samples in both herds than did the complement fixation test. Following cloning, the isolates of M. capricolum subsp. capripneumoniae were analysed biochemically and shown to be metabolically similar. They oxidized glucose, N-acetylglucosamine, pyruvate and L-lactate with high affinity and mannose, glucosamine and 2-oxobutyrate with low affinity; they were unable to utilize maltose, trehalose, fructose or ethanol. Major improvements were seen in the growth yield of the Eritrean strains with the addition of pyruvate to the medium. Thus, it may be that organic acids are important energy sources for these strains and may be used in addition to or in place of glucose. In contrast to most other strains of the M. mycoides cluster, the Eritrean strains produced large amounts of hydrogen peroxide during the oxidation of NADH by lysed cells. This characteristic had previously been reported for strain M. F38, the type strain of M. capricolum subsp. capripneumoniae, although strain F38 did not metabolize sugars. Hydrogen peroxide has long been considered a pathogenicity factor in mycoplasma infections. This is the first isolation of M. capricolum subsp. capripneumoniae from Eritrea.

Differential inhibition of mollicute growth: an approach to development of selective media for specific mollicutes.

The energy-generating pathways of Mycoplasma spp. are diverse. Thus, it was predicted that the ability of inhibitors of these pathways to block growth would vary among species. This prediction was tested with 14 Mycoplasma species and potential inhibitors. The greatest differentiation among test species was obtained using fluoride, iodoacetate (IAA), beta-fluoropyruvate (FP), cibacron blue (CB), L-citrulline, and carbonyl cyanide m-chlorophenylhydrazone. A range of other potential inhibitors, including L-arginine analogues, had little inhibitory effect on growth, and D-arginine was shown to be a growth substrate for arginine-hydrolyzing species. Fluoride selectively inhibited the growth of mycoplasmas that were able only to ferment sugars to lactate and/or to hydrolyze arginine. In contrast, IAA was most effective against organic acid-oxidizing species, and L-citrulline inhibited arginine-hydrolyzing species. Mycoplasma verecundum, a species for which energy sources have not been identified, was relatively resistant to FP. Similarly, Acholeplasma laidlawii was distinguished by its CB resistance.

New chromogenic agar medium for the identification of Candida spp.

A new chromogenic agar medium (Candida diagnostic agar [CDA]) for differentiation of Candida spp. is described. This medium is based on Sabouraud dextrose agar (Oxoid CM41) and contains (per liter) 40.0 g of glucose, 10.0 g of mycological peptone, and 15.0 g of agar along with a novel chromogenic glucosaminidase substrate, ammonium 4-(2-[4-(2-acetamido-2-deoxy-beta-D-glucopyranosyloxy)-3-methoxyphenyl]-vinyl)-1-(propan-3-yl-oate)-quinolium bromide (0.32 g liter(-1)). The glucosaminidase substrate in CDA was hydrolyzed by Candida albicans and Candida dubliniensis, yielding white colonies with deep-red spots on a yellow transparent background after 24 to 48 h of incubation at 37 degrees C. Colonies of Candida tropicalis and Candida kefyr were uniformly pink, and colonies of other Candida spp., including Candida glabrata and Candida parapsilosis, were white. CDA was evaluated by using 115 test strains of Candida spp. and other clinically important yeasts and was compared with two commercially available chromogenic agars (Candida ID agar [bioMerieux] and CHROMagar Candida [CHROMagar Company Ltd.]). On all three agars, colonies of C. albicans were not distinguished from colonies of C. dubliniensis. However, for the group containing C. albicans plus C. dubliniensis, both the sensitivity and the specificity of detection when CDA was used were 100%, compared with values of 97.6 and 100%, respectively, with CHROMagar Candida and 100 and 96.8%, respectively, with Candida ID agar. In addition, for the group containing C. tropicalis plus C. kefyr, the sensitivity and specificity of detection when CDA was used were also 100%, compared with 72.7 and 98.1%, respectively, with CHROMagar Candida. Candida ID agar did not differentiate C. tropicalis and C. kefyr strains but did differentiate members of a broader group (C. tropicalis, C. kefyr, Candida lusitaniae plus Candida guilliermondii); the sensitivity and specificity of detection for members of this group were 94.7 and 93.8%, respectively. In addition to the increased sensitivity and/or specificity of Candida detection when CDA was used, differentiation of colony types on CDA (red spotted, pink, or no color) was unambiguous and did not require precise assessment of colony color.

Orotrachial intubation in darkness using night vision goggles.

OBJECTIVE: Securing the airway of a wounded soldier while operating in a light-restricted combat environment may be required of forward-deployed military medical personnel. The best method of obtaining such an airway has not been addressed. In this pilot study, the objective was to examine the use of endotracheal intubation using an infrared filtered laryngoscope and night vision goggles. METHODS: The investigators performed endotracheal intubation, using an infrared filter light source laryngoscope, on patients undergoing elective surgical procedures. All intubations took place in a completely darkened operating room. RESULTS: Twenty-one patients (91.3%) were intubated successfully as defined in the study. No adverse outcomes or complications occurred. CONCLUSIONS: This study demonstrates that endotracheal intubation can be performed using a laryngoscope with an infrared filter and night vision goggles with a high success rate in a select population in a darkened environment.

Rapid screening of H(2)O(2) production by Mycoplasma mycoides and differentiation of European subsp. mycoides SC (small colony) isolates.

Mycoplasma mycoides strains were screened for the ability to produce H(2)O(2) from glucose and glycerol metabolism using rapid and simple colorimetric assays. In quantitative assays, H(2)O(2) production by washed cell suspensions was detected by the oxidation of o-dianisidine in the presence of peroxidase. In qualitative assays, a 3,3'-diaminobenzidine-peroxidase reagent was applied to colonies on agar plates. Both methods enabled differentiation of European subsp. mycoides SC (small colony) isolates from other M. mycoides strains by their inability to produce H(2)O(2) from glycerol metabolism. In addition, two strains of subsp. capri were identified which produced large amounts of H(2)O(2) from glucose oxidation. In lysed cells of these strains, NADH oxidation gave approximately 1 mol H(2)O(2) per mol NADH oxidised whereas in 36 subsp. mycoides and 10 other subsp. capri strains, the quantity produced was 0.01-0.20mol H(2)O(2) per mol NADH oxidised.

Biochemical characterization of Mycoplasma bovirhinis, Mycoplasma dispar and recent bovine isolates of Mycoplasma canis.

The pattern and kinetics of substrate utilization by the type strains of Mycoplasma canis, M. bovirhinis and M. dispar and ten recent M. canis isolates from cattle were determined. Metabolism of a range of sugars and organic acids by M. dispar was detectable by measurement of oxygen uptake. Organic acids were not utilized by M. bovirhinis or M. canis, and there was no oxygen uptake during metabolism of glucose or other sugars, as monitored by a pH-change method. The M. canis strains varied in their ability to metabolize sugars; seven of the isolates from cattle had the distinctive ability to metabolize sucrose, and one isolate, plus the type strain (from a dog), metabolized N-acetylglucosamine. The M. bovirhinis strain metabolized maltose. However, all the test strains oxidized glycerol at high rates and with a high affinity. Oxidation of glycerol has been reported for other mycoplasmas from the bovine respiratory tract and leads to the production of hydrogen peroxide, a potential virulence factor.

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 (gamma-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.

A rapid biochemical test to aid identification of Mycoplasma mycoides subsp. mycoides small colony (SC) strains.

The ability to utilize maltose, as determined by measurement of oxygen uptake, is used to differentiate Mycoplasma mycoides subsp. mycoides small colony (SC) and M. capricolum subsp. capripneumoniae (all strains negative) from other members of the M. mycoides cluster (M. mycoides subsp. capri, M. mycoides subsp. mycoides large colony (LC), M. capricolum subsp. capricolum; and bovine serogroup 7; 94% of strains positive). Rapid tests for maltose utilizing ability were developed, based on hydrolysis of a chromogenic alpha-glucosidase (maltase) substrate (p-nitrophenyl-alpha-D-glucopyranoside, colourless) to give a brightly coloured product (p-nitrophenol, yellow). On agar plates, colonies of maltose-utilizing strains became coloured within 40 min.

Kinetics and distribution of alcohol oxidising activity in Acholeplasma and Mycoplasma species.

Alcohol metabolism by Acholeplasma and Mycoplasma cell suspensions was determined using changes in dissolved oxygen tension to monitor oxygen uptake. All seven Acholeplasma test species oxidised ethanol and (where tested) propanol, butanol and pentanol. The rate of oxidation, at any particular substrate concentration, decreased with increasing alcohol molecular mass. Amongst 20 Mycoplasma species tested, M. agalactiae, M. bovis, M. dispar, M. gallisepticum, M. pneumoniae and M. ovipneumoniae oxidised ethanol. Propanol was also oxidised by M. dispar and isopropanol by M. agalactiae, M. bovis and M. ovipneumoniae. Isopropanol was oxidised at particularly high rates (V(max)100 nmol O(2) taken up min(-1) mg cell protein(-1)) and with a relatively high affinity (K(m) value<2 mM); oxygen uptake was consistent with oxidation to acetone. The significance of alcohol oxidation is unclear, as it would not be predicted to lead to ATP synthesis.

A novel chromogenic ester agar medium for detection of Salmonellae.

A novel agar medium, chromogenic Salmonella esterase (CSE) agar, for the differentiation of salmonellae is described. The agar contains peptones and nutrient extracts together with the following (grams per liter unless otherwise specified): 4-[2-(4-octanoyloxy-3, 5-dimethoxyphenyl)-vinyl]-quinolinium-1-(propan-3-yl carboxylic acid) bromide (SLPA-octanoate; bromide form), 0.3223; lactose, 14. 65; trisodium citrate dihydrate, 0.5; Tween 20, 3.0; ethyl 4-dimethylaminobenzoate, 0.035% (wt/vol), novobiocin, 70 mg liter-1. The key component of the medium is SLPA-octanoate, a newly synthesized ester formed from a C8 fatty acid and a phenolic chromophore. In CSE agar, the ester is hydrolyzed by Salmonella spp. to yield a brightly colored phenol which remains tightly bound within colonies. After 24 h of incubation at 37 or 42 degreesC, colonies of typical Salmonella spp. were burgundy colored on a transparent yellow background, whereas non-Salmonella spp. were white, cream, yellow or transparent. CSE agar was evaluated by using a panel of strains including a high proportion of Salmonella and non-Salmonella strains giving atypical reactions on other differential agars. The sensitivity (93.1%) of CSE agar for non-typhi salmonellae compared favorably with those of Rambach (82. 8%), xylose-lysine-deoxycholate (XLD; 91.4%), Hektoen-enteric (89.7%), and SM ID (91.4%) agars. The specificity (93.9%) was also comparable to those of other Salmonella media (SM ID agar, 95.9%; Rambach agar, 91.8%; XLD agar, 91.8%; Hektoen-enteric agar, 87.8%). Strains of Citrobacter freundii and Proteus spp. giving false-positive reactions with other media gave a negative color reaction on CSE agar. CSE agar enabled the detection of >30 Salmonella serotypes, including agona, anatum, enteritidis, hadar, heidelberg, infantis, montevideo, thompson, typhimurium, and virchow, which accounted for 91.8% of the salmonella isolates recorded by the Public Health Laboratory Service (Colindale, London, England) for 1997.

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.