Phenotypic switching of antibiotic resistance circumvents permanent costs in Staphylococcus aureus.

Bacterial antibiotic resistance is often associated with a fitness cost in the absence of the antibiotic [1,2]. We have examined a resistance mechanism in Staphylococcus aureus that negates these costs. Exposure to gentamicin both in vitro and in vivo has been reported to result in the emergence of a gentamicin-resistant small colony variant (SCV)[3-8]. We show that the emergence of SCVs following exposure to gentamicin results from a rapid switch and that bacteria exposed to cycles of gentamicin followed by antibiotic-free medium repeatedly switched between a resistant SCV and a sensitive parental phenotype (revertants). The fitness of revertants relative to S. aureus with stable gentamicin resistance was greater in drug-free media, which suggests that S. aureus has evolved an inducible and reversible resistance mechanism that circumvents a permanent cost to fitness.

Environmentally constrained mutation and adaptive evolution in Salmonella.

The relationship between environment and mutation is complex [1]. Claims of Lamarkian mutation [2] have proved unfounded [3-5]; it is apparent, however, that the external environment can influence the generation of heritable variation, through either direct effects on DNA sequence [6] or DNA maintenance and copying mechanisms [7-10], or as a consequence of evolutionary processes [11-16]. The spectrum of mutational events subject to environmental influence is unknown [6] and precisely how environmental signals modulate mutation is unclear. Evidence from bacteria suggests that a transient recombination-dependent hypermutational state can be induced by starvation [5]. It is also apparent that changes in the mutability of specific loci can be influenced by alterations in DNA topology [10,17]. Here we describe a remarkable instance of adaptive evolution in Salmonella which is caused by a mutation that occurs in intermediate-strength osmotic environments. We show that the mutation is not 'directed' and describe its genetic basis. We also present compelling evidence in support of the hypothesis that the mutational event is constrained by signals transmitted from the external environment via changes in the activity of DNA gyrase.

The mutagenic properties of N-nitrosopeptides in the Ames test.

N-(N-Acetyl-L-prolyl)-N-nitrosoglycine (APNG) and N-(N-acetylvalyl)-N-nitrosoglycine (AVNG) are shown to exert mutagenic activity in the Salmonella/mammalian microsome mutagenicity (Ames) test. Positive responses are apparent for base-pair substitution mutation-detecting strains (TA1535, TA100 and TA102) both with and without the addition of S9-mix. It is concluded that both APNG and AVNG are direct-acting mutagens.

An investigation of the endogenous formation of apparent total N-nitroso compounds in conventional microflora and germ-free rats.

The endogenous formation of apparent total N-nitroso compounds (ATNC) has been investigated in germ-free (GF) and conventional (CV) microflora rats as a function of the drinking-water nitrate concentration. ATNC levels were below the 40 micrograms (N-NO)/kg detection limit in the blood, liver, kidney, spleen and small intestine of all CV and GF rats. For the CV rats ATNC were detected in concentrations of up to 370 micrograms (N-NO)/kg in the large intestine and up to 50 micrograms (N-NO)/kg in the stomach and there was a significant positive correlation between ATNC formation and the drinking-water nitrate level. Comparison of these results with those from GF rats showed that the ATNC in the stomach and large intestine of the CV animals were formed by microbial action, most probably involving bacterial nitrate-reductase activity.

Aluminium uptake from some foods by guinea pigs and the characterization of aluminium in in vivo intestinal digesta by SEC-ICP-MS.

The uptake of ingested aluminium (Al) from food items commonly consumed in a normal human diet was investigated by feeding five test diets to guinea pigs. Al concentrations were measured in the femur, brain, kidney and upper intestinal contents. Consumption of these diets did not lead to elevated Al levels in brain. Levels of Al in the bone were elevated in animals fed sponge cake with a permitted Al-containing additive, and the presence of citrate as orange juice enhanced bone deposition and increased kidney Al levels. Less than 1% of Al in the upper intestinal contents was found in the soluble fraction, and characterization by SEC-ICP-MS indicated that this Al was not present as Al-citrate.

A study of the toxicity of five mineral hydrocarbon waxes and oils in the F344 rat, with histological examination and tissue-specific chemical characterisation of accumulated hydrocarbon material.

Five food-grade mineral hydrocarbon (MHC) materials; a low melting point wax (LMPW), a synthetic wax (C80W) and three white oils (N15H, N70H and P70H) were administered orally to female Fischer-344 rats for 28 and 90 days at a dose level of 2% in the diet. Tissues were examined at autopsy for any treatment-related histopathological changes. The histology of target organs was the same as found in previous studies on LMPW and mineral oils and similar effects were also observed from feeding C80W. Chemical analysis showed no detectable levels of MHCs in urine and no discernible differences in the MHC profile in faeces extracts compared to diets. The presence of MHCs in most tissues was not always associated with observable histological changes. The notable observations were MHC material was detected in all tissues of rats fed with diets containing LMPW and C80W. The levels found ranged from 0.04 to 1.52% by weight for the LMPW and from 0.01 to 0.75% for the C80W. MHC material was detected in all samples of small intestine, heart and kidney for all groups. Only the livers from rats administered with LMPW and C80W were analysed, which were found to contain MHC material. Preferential accumulation of MHCs was in the alkane range approximately C(20)-C(35). The findings indicate that the size and the structure of individual components play a role both in determining their propensity to accumulate in different tissues and in the severity of any response that they elicit once they have accumulated. The implication of these findings are discussed in the context of specifications for 'food-grade' mineral hydrocarbons such as used as food additives. The data presented here suggests that the current specifications are not prescriptively adequate in controlling the amount of MHC material between C(25) and C(35) that can accumulate.

Analysis of polycyclic aromatic hydrocarbons in UK total diets.

Analysis of UK total-diet samples for polycyclic aromatic hydrocarbons was carried out using a simplified sample clean-up and a high-performance liquid chromatography dual fluorescence detector system. The results indicate that cereals and oils/fats contribute the major part (approximately one third each) of the polycyclic aromatic hydrocarbons in these total diets. Fruit, sugars and vegetables provide much of the remainder (approximately one quarter) while meat, fish, milk and beverages make relatively minor contributions. These results are compared with others in the current literature on polycyclic aromatic hydrocarbons in foods. The levels in the UK diet seem to be at least as low as those found elsewhere.

N-nitroso compounds and polycyclic aromatic hydrocarbons in Icelandic smoked cured mutton.

Five samples of Icelandic smoked cured mutton were analysed for volatile N-nitrosamines, N- nitrosoamino acids, total N-nitroso content and several polycyclic aromatic hydrocarbons. Levels varied considerably from sample to sample but generally the levels of polycyclic aromatic hydrocarbons and N-nitrosamines (as judged by N- nitrosoproline , N- nitrosohydroxyproline and total N-nitroso content) varied in the same way. It is suggested that in this product the smoking procedure is responsible for both groups of contaminants and that the curing process is relatively unimportant.

Mutagenic properties of N-nitrosopeptides in mammalian cell culture assays.

Two N-nitrosopeptides, N-(N-acetyl-L-prolyl)-N-nitrosoglycine and N-(N-acetylvalyl)-N-nitrosoglycine, were investigated for genetic toxicity towards mammalian cells using an established line of Chinese hamster ovary cells (CHO-K1-BH4). Observations were made on three indices of genetic damage, namely chromosome aberrations, sister chromatid exchange and induction of thioguanine-resistant variants. Treatment of cells with either compound resulted in dose-dependent increases in all indices, indicating that both compounds are direct-acting mutagens.

Molecular mechanisms of Staphylococcus aureus nasopharyngeal colonization.

Staphylococcus aureus is responsible for a wide range of different infections ranging in severity from mild to fatal. However, it primarily exists as a commensal organism in a number of different anatomical sites including the nasopharynx. Although colonization itself is a harmless state, colonized individuals are at risk of endogenous infection when S. aureus enters otherwise sterile sites via wounds or indwelling medical devices. As such, studies of colonization may identify important targets for vaccines or other prophylactic approaches. Colonization is a dynamic process; S. aureus must attach to host surfaces, overcome immune components and compete with other commensal microbes. This occurs via a number of surface-attached and secreted proteins and other factors such as wall teichoic acid. In addition, colonizing S. aureus must constantly replicate to maintain its niche and exclude other strains. These myriad interactions provide a strong selective pressure for the maintenance or enhancement of mechanisms of adhesion, invasion and immune evasion. The evolutionary implications of this may explain why S. aureus is such a capable pathogen because many of the proteins involved in colonization have also been identified as virulence factors. This review describes the diverse molecular mechanisms used by S. aureus to colonize the host and discusses how the pressures that have selected for these may have led to its virulence.

Surveillance of preservatives and their interactions in foodstuffs.

The organization of food surveillance in the UK is described, in particular as it has been applied to preservatives and their interaction products in foods. Applications of nitrates and nitrites as preservatives are discussed, together with the consequential exposure of consumers to these anions and their reaction products. Analytical methods for the determination of volatile and non-volatile N-nitroso compounds are referred to in relation to the results in food surveillance studies. Concentrations of Apparent Total N-nitroso Compounds (ATNC) averaged 2900 micrograms(N-NO)/kg in fried smoked bacon compared with 2400 micrograms/kg in fried unsmoked bacon; of this, known volatile and non-volatile N-nitroso compounds accounted for only 10-20%. ATNC were not detected in cheeses except those manufactured with added nitrate when ATNC levels up to 210 micrograms(N-NO)/kg were detected. Further studies are needed to determine the identity and toxicological properties of the non-volatile N-nitroso compounds.

The formation and occurrence of amino acid pyrolysates and related mutagens in cooked foods.

The classes of cooked foods that contain detectable levels of mutagenic activity are discussed together with the effects of different cooking procedures on the extent of mutagen formation. Analytical procedures that have so far been devised to quantify the concentrations of specific mutagenic compounds are described and the levels of these species that have been detected in cooked foods are detailed.

A method for the investigation of free and protein-bound N-nitrosoproline in beer.

Hydrolysis of beer peptides and proteins by both mild enzymatic and vigorous alkaline conditions has established that peptide-bound N-nitrosoproline residues are not present in beer in significant quantities. Free N-nitrosoproline remains the single most abundant identified N-nitroso compound, and this accounts for up to 10% of the apparent total N-nitroso compounds (ATNC). Both hydrolysis and extractability studies strongly indicate that the majority of the ATNC are not associated with beer peptides, but rather are likely to be low molecular weight possessing in some cases an acidic functional group.

Isotope ratio and isotope dilution analysis of lead in wine by inductively coupled plasma-mass spectrometry.

The lead content of 12 wine samples was measured by inductively coupled plasma-mass spectrometry using the methods of standard addition and isotopic dilution analysis. An additional wine sample was analysed by external calibration, standard addition and isotopic dilution analysis. The lead content of the wine samples was in the range 30-150 ng ml-1 and good agreement between the different techniques was observed. Analysis of lead isotope ratios revealed significant differences between Australian and European wines, reflecting the different isotopic composition of Australian lead.

The aluminium content of infant formula and tea.

Tea and infant formulae have been examined for the presence of aluminium. Concentrations in 13 different tea infusions ranged from 2.2 mg/l to 4.5 mg/l. In cow's milk-based infant formulae as made up for consumption, aluminium levels of between 0.03 mg/l and 0.20 mg/l were detected. Higher amounts were present in soya-based formulae with concentrations ranging from 0.64 mg/l to 1.34 mg/l.

The collaborative evaluation of a procedure for the determination of N-nitroso compounds as a group.

Two collaborative studies have been made of the method devized for the determination of N-nitroso compounds as a group, which involves their selective denitrosation with hydrogen bromide in glacial acetic acid, followed by the measurement of the nitric oxide liberated using a chemiluminescence analyzer, such as the TEA. Considerable variation was evident between the results in the first study, most of the values reported being low. This situation undoubtedly arose from the carry-over of traces of hydrogen bromide from one determination to the next, which resulted in premature denitrosation. The precision and consistency of the results were greatly improved in the second study, for which additional precautions had been recommended. All of the six participating laboratories reported reasonably consistent values, the coefficients of variation being 27.9% and 21.1% respectively, at the levels of spiking of N-nitrososarcosine equivalent to 32.9 ng and 146.4 ng of nitric oxide.

Measurement of haloacetic acids and their decomposition in wine by capillary gas chromatography.

A method has been developed for the analysis of haloacetic acids in wine involving solid-phase extraction followed by methylation and quantification by capillary gas chromatography with electron capture detection. Recoveries from spiked samples were greater than 85% for each of the acids and the limits of detection of the method were 100, 10 and 5 micrograms/litre for the chloroacetic, bromoacetic and iodoacetic acids, respectively. The haloacetic acids were not detected in a survey of over 30 retail samples. Separate stability studies showed that the concentration of chloroacetic acid added to wine was essentially unaffected by up to 90 days storage at 30 degrees C. Under the same conditions, however, less than 20% of bromoacetic acid and iodoacetic acid remained.

The effect of oxygen on nitrosamine formation in bacon.

Bacon has been cooked in air and in nitrogen atmospheres and the effect on the formation of N-nitrosodimethylamine and N-nitrosopyrrolidine has been studied. Under nitrogen, reductions of 50%-90% were obtained in the concentrations of both nitrosamines in the cooking vapour. The results are discussed in terms of the likely role of nitric oxide in nitrosamine formation in cooking bacon.

Factors affecting the polycyclic aromatic hydrocarbon content of cereals, fats and other food products.

Factors affecting polycyclic aromatic hydrocarbon (PAH) concentrations in oils and fats, cereals and related foodstuffs have been investigated. Levels of PAHs were low in retail fish and animal-derived oils and fats, such as butter, where the mean benzo(a)pyrene concentration was 0.06 microgram/kg. Higher and more variable amounts were present in retail vegetable oils for which the mean level of benzo(a)pyrene was 1.29 micrograms/kg. Margarine was the major dietary source of PAHs in the oils and fats total diet group accounting for 70% of the benzo(a)pyrene intake from these commodities. The levels of benzo(a)pyrene were less than 0.1 microgram/kg in white flour and similar amounts were found in bread showing that PAHs are not formed to any significant extent during baking of bread. Higher concentrations of up to 2.2 micrograms/kg benzo(a)pyrene were detected in cereal-derived products containing higher levels of edible oils such as pudding-based desserts, biscuits and cakes. The presence of vegetable oils as an ingredient also appeared to increase PAH levels in infant formulae as the mean benzo(a)pyrene content of 0.49 microgram/kg was four times higher than that found in skimmed milk. The mean value in the feed, after reconstituting the formulae with water, would however have been less than 0.1 microgram/litre. Investigations of rape seed drying showed no increase in any PAHs when cold, or electrically-heated air was used. Combustion gas drying had no effect for the larger PAHs such as benzo(a)pyrene but caused mean increases of between 41% and 126% for fluoranthene, pyrene and chrysene. These increases did not correlate with reductions in moisture content of the rape seed implying that the combustion conditions were more important to PAH contamination than the degree of exposure to combustion gases. Concentrations of these three PAHs and also benz(a)anthracene were all significantly reduced by up to a factor of five when crude oils were refined suggesting that carefully controlled direct drying need not contribute PAHs to refined oils and fats.

An investigation of apparent total N-nitroso compounds in beer.

The concentration of apparent total N-nitroso compounds (ATNC) in beer has been investigated using a group-selective procedure based on chemical denitrosation with hydrogen bromide and chemiluminescence detection of the released nitric oxide. In a survey of samples of 40 brands of beer and lager, detectable levels of ATNC were present in 17 samples at concentrations of 20-100 micrograms N-NO/kg in 11 and 100-500 micrograms N-NO/kg in six. To determine the origin of ATNC in beer the production of a commercial batch was examined in detail. ATNC levels were below the detection limit in the sweet wort (aqueous extract of malt), bitter wort (malt extract boiled with hops) and also at the start of fermentation, but during the course of fermentation the concentration of ATNC increased appreciably and that of inorganic nitrate decreased; detectable, though transitory, levels of inorganic nitrite were observed. None of the brewing ingredients contained sufficiently high enough levels of ATNC to account for the concentration of these compounds present in the beer after fermentation. These findings suggest that the presence of detectable levels of ATNC in some beers is a result of N-nitrosation reactions occurring in the fermenting wort with the nitrosating species derived from reduction of nitrate, due probably to the presence of microbial species with nitrate reductase activity.