Evaluation of the Vitek Immunodiagnostic Assay System (VIDAS) for the detection of Salmonella in foods

A Salmonella Assay using the Vitek Immunodiagnostic Assay System (VIDAS) was compared with a conventional cultural method (CCM) for the detection of salmonellas in 141 samples of artificially and naturally contaminated foods. There was an overall agreement of 92.9% between the methods. The productivity of the VIDAS Salmonella Assay (VSA) was not improved using an alternative enrichment protocol for the detection of Salmonella in 12 raw meat samples.
The sensitivity and specificity of the VSA was assessed using pure cultures of salmonellas and non-salmonellas. The detection limit was 1.8 times 10⁶ salmonellas ml^-1 in M-broth and some Citrobacter freundii strains gave false-positive results.
Using an immunomagnetic separation (IMS) technique and an abbreviated cultural enrichment, the VSA results could be obtained a day earlier than the standard VSA method.     

OPTIMIZATION OF CULTURE CONDITIONS FOR ENUMERATION OF H2S BACTERIA IN THE FLESH OF SEAFISH

H₂S bacteria of seafish flesh are weakly halophilic and require on average 1.68% NaCl according to statistical studies. Enumeration is optimal on PCA-H₂S(a PCA medium supplemented with sulfur sources and increased NaCl concentrations) incubated at 25C. Total aerobic bacteria can be counted simultaneously on this medium. The proportion of H₂S bacteria relative to total aerobic bacteria increased slightly during prolonged storage of the fish, but was highly variable. Models relating H₂S bacterial counts to spoilage of fish are sigmoidal and showed that when the count exceeds 10,000 CFU/g, whole or filleted fish stored in ice at 0C are unfit for consumption. Shewanella putrefaciens accounted for 69% of the H₂S bacteria at the fifth day of storage and 100% at the fifteenth.     

Hydrogen sulfide and the vasculature: a novel vasculoprotective entity and regulator of nitric oxide bioavailability?

Hydrogen sulfide (H₂S) is a well known and pungent toxic gas that has recently been shown to be synthesised in man from the amino acids cystathionine, homocysteine and cysteine by at least two distinct enzymes; cystathionine-γ-lyase and cystathionine-β-synthase. In the past few years, H₂S has emerged as a novel and increasingly important mediator in the cardiovascular system but delineating the precise physiology and pathophysiology of H₂S is proving to be complex and difficult to unravel with disparate findings reported with cell types, tissue types and animal species reported. Therefore, in this review we summarize the mechanisms by which H₂S has been proposed to regulate blood pressure and cardiac function, discuss the mechanistic discrepancies reported in the literature as well as the therapeutic potential of H₂S. We also examine the methods of H₂S detection in biological fluids, processes for H₂S removal and discuss the reported blood levels of H₂S in man and animal models of cardiovascular pathology. We also highlight the complex interaction of H₂S with nitric oxide in regulating cardiovascular function in health and disease.     

Plant responses to H2S and SO2 fumigation. II. Differences in metabolism of H2S and SO2 in spinach

Fumigation of spinach (Spinacia oleracea L. cvs Estivato and Monosa) with H₂S or SO, for 1 to 6 days resulted in accumulation of sulfhydryl (SH) compounds in the shoots of both H₂S- and SO₂-exposed plants. The sulfate concentration in shoots of SO₂-exposed plants increased linearly with time. SH accumulation showed saturation kinetics as a function of time as well as H₂S concentration, ascribed to the internal H₂S concentration in the plant and the availability of substrates for glutathione synthesis, respectively. SH compounds accumulated more at lower exposure temperatures, whereas sulfate accumulation was more pronounced at higher temperatures. These results are discussed in relation to the possible foliar uptake of H₂S and SO₂, the temperature dependence of uptake and the water solubility of these gases. The possibility of SO₂-induced H₂S emission rather than sulfate accumulation as a source for SH accumulation is also discussed. Cessation of fumigation resulted in a decrease in SH compounds and sulfate content that could be accounted for by sulfur metabolism and growth, respectively.     

Use of impedance measurements to estimate numbers of antibiotic resistant Salmonella strains

Impedance detection times were compared with traditional plating methods for enumerating antibiotic-resistant strains of Salmonella stanley, Salm. thompson and Salm. infantis grown in laboratory medium and pork slurry. The correleation of log₁₀ counts of salmonellas with detection times was highly significant ( r = -0·96 for broth and r = -0·94 for slurries. The confidence limits (± og₁₀ 1·0 for broth and ± log₁₀ 1·65 for slurry) indicated that detection times could reliably be used as a rapid means of enumerating salmonellas when large numbers of counts of known strains are required for growth studies. Use of antibiotic-resistant strains also permitted their selective detection by impedance from the natural spoilage flora of pork slurry when the same antibiotics were incorporated in the detection medium.     

Isotope effects associated with the anaerobic oxidation of sulfide by the purple photosynthetic bacterium, Chromatium vinosum

Abstract Small inverse isotope effects of 1–3‰ were consistently observed for the oxidation of sulfide to elemental sulfur during anaerobic photometabolism by Chromatium vinosum . The inverse fractionation can be accounted for by an equilibrium isotope effect between H₂S and HS⁻, and may indicate that C. vinosum (and other photosynthetic bacteria) utilizes H₂S rather than HS⁻ as the substrate during sulfide oxidation.     

The effect of short-term H2S fumigation on water-soluble sulphydryl and glutathione levels in spinach

Abstract. Short-term fumigation of Spinacia oleracea with 380 μg m⁻³ H₂S (250 ppb) resulted in a rapid accumulation of water-soluble SH-compounds in the shoots. After 1 h exposure a substantial increase in the SH-content was already detectable and maximal accumulation, three- to four-fold that in control plants, was observed after 24 h of exposure. Irradiation during H₂S exposure only slightly affected the rate and level of SH-accumulation. H₂S fumigation did not affect the water-soluble SH-content of the roots. Glutathione was the sole water-soluble SH-compound accumulating upon exposure to H₂S. It was calculated that during the first hour of exposure to 380 μg m⁻³ H₂S 39% of the possible absorbed H₂S was converted into glutathione. The SH-content of the water-soluble proteins of the shoots was not affected by H₂S exposure. When fumigation was stopped, a rapid decrease in glutathione content was observed and after 48 h the content was comparable to that of the control plants. Contrary to H₂S, SO₂ fumigation did not result in a rapid accumulation of glutathione in spinach shoots. The possible role of glutathione accumulation during H₂S fumigation is discussed.     

The microplankton organisms at the oxic-anoxic interface in the pelagial of the Black Sea

Abstract The semi-permanent anoxia in the deep-waters of the Black Sea supported the formation of a unique microplankton community at the oxicanoxic interface. One group of ciliates ( Pleuronema marinum, Askenasia sp., species of the families Tracheliidae, Holophryidae and Amphileptidae) inhabited the water layer just above the upper boundary of H₂S, and was spatially associated with and fed on large colorless sulphur bacteria ( Thiovulum spp.). Other ciliates (species of order Scuticociliatida) populated the upper layer of the H₂S zone. A significant part of them possessed ectosymbiotic bacteria. Since the metazoans were not found in the O₂ and H₂S boundary layer, the protist community is considered to be the main factor in utilization of chemotrophic bacterial production.     

Cysteine-induced H2S emission, ATP depletion, and membrane electrical responses in oat coleoptiles

Incubation of oat ( Avena sativa L. cv. Victory) coleoptile segments in 4 m M L-cysteine reduced the tissue ATP level to 42 nmol ( g fresh weight)⁻¹ (35% of normal) over a 2 h period. Emissions of H₂S accompanied this depletion in ATP suggesting an H₂S production by desulfhydration of cysteine similar to that reported in other plants. Additions of exogenous H₂S to the sections also caused ATP depletion. Aminooxyacetate, an inhibitor of cysteine desulfhydrase (EC 4.4.1.1), eliminated the cysteine-induced H₂S emission and the ATP depletion. Prolonged exposure to cysteine depressed the electrical polarity of the cell membrane from – 116 mV to –85 mV. That and other electrical responses appear to reflect a reduced capacity for ATP-dependent H⁺ extrusion. These effects should be taken into account whenever cysteine is used in physiological experiments.     

A study of isolation procedures for multiple infections of Salmonella and Arizona in a wild marsupial, the quokka (Setonix brachyurus)

Rectal swabs and faeces were used in the regular sampling for salmonellas and Arizonas from a heavily-infected population of a marsupial, the quokka ( Setonix brachyurus ). The media used were strontium selenite A and strontium chloride B enrichment broths, with subculture onto modified bismuth sulphite agar and deoxycholate citrate agar. A study of sampling, enrichment, sub-culture and colony selection procedures produced an optimal scheme giving high yields but consistent with reasonable economy of time and materials. A three-swab sample was taken and inoculated into the two enrichment media, and with each enrichment subjected to three subcultures. The absolute efficiency of this procedure was greater than 80% (and confirmed by a serological method), compared with only 67% for a single swab in a single enrichment. Recovery of some serotypes depended on the media used; e.g. Arizonas could not be recovered satisfactorily from strontium chloride B enrichment. Faeces samples were found to be greatly superior to rectal swabs for detecting salmonellas and arizonas but they were less convenient in field studies. In a comparison of rectal swabs and faeces samples where the actual concentration of salmonellas was known, it was found that the efficiency of rectal swabs approached 100% if there were more than 10³ salmonellas/g faeces, but this declined to approximately 50% if there were 10²-10³ salmonellas/g faeces and only 25% if there were less than 10² salmonellas/g faeces. A new statistical procedure was introduced for comparing the number of isolations from two methods, and this should be of use in similar methodological studies.     

Production of the gaseous signal molecule hydrogen sulfide in mouse tissues

The gaseous molecule hydrogen sulfide (H₂S) has been proposed as an endogenous signal molecule and neuromodulator in mammals. Using a newly developed method, we report here for the first time the ability of intact and living brain and colonic tissue in the mouse to generate and release H₂S. This production occurs through the activity of two enzymes, cystathionine-γ-lyase and cystathionine-β-synthase. The quantitative expression of messenger RNA and protein localization for both enzymes are described in the liver, brain, and colon. Expression levels of the enzymes vary between tissues and are differentially distributed. The observation that, tissues that respond to exogenously applied H₂S can endogenously generate the gas, strongly supports its role as an endogenous signal molecule.     

A note on the effect of the lactoperoxidase systems on salmonellas in vitro and in vivo

The lactoperoxidase system (LPS), a natural bactericidal system in milk, was investigated for its activity against salmonellas in vivo and in vitro. In acidified raw milk, in which the LPS was supplemented with an exogenous supply of H₂O₂, the numbers of salmonellas decreased rapidly. Different salmonella serotypes were affected to the same extent; rough strains, however, were more susceptible than smooth strains. When calves were fed on fresh milk, containing the LPS, and challenged with Salmonella typhimurium in doses of either 10⁹ or 10¹⁰, the clinical findings and salmonella excretion patterns were similar to those of control calves fed on heated milk. It was concluded that further studies, perhaps in the field, are necessary to evaluate LPS as a possible non-antibiotic system to control salmonellosis.     

A Note on the Inhibition of Pseudomonas aeruginosa by a Modification of Brilliant Green Agar for Improved Salmonella Isolation

A strain of Pseudomonas aeruginosa having colonies that resemble those of salmonellas on brilliant green agar is almost totally inhibited by the addition of 1.0 mg/ml of sulphacetamide to the medium. Low numbers of Ps. aeruginosa grew equally well on brilliant green and nutrient agar, but 10⁶–10⁷ organisms were needed before any growth appeared on the medium containing sulphacetamide. During 12 months of routine use of the sulphacetamide medium, involving almost 3000 plates, Ps. aeruginosa has been isolated as a contaminant only once. Forty-seven salmonella serotypes were grown on the sulphacetamide brilliant green agar in the same period.     

A TRIPLE SUGAR-UREA-IRON MEDIUM FOR THE CONFIRMATION OF SALMONELLA AND THE EXCLUSION OF PROTEUS STRAINS

SUMMARY: A modification of the dulcitol-lactose-iron medium of Taylor & Silliker (1958) is proposed. It contains sucrose as well as lactose to aid in detecting slow lactose fermenters and gives simultaneously tests for urea hydrolysis and H₂S production. The latter reaction is kept apart from the carbohydrate fermentations and the urea hydrolysis. The surface growth can be used in a test for the deamination of phenylalanine to phenylpyruvic acid.     

Effect of carbohydrate source in selenite cystine trimethylamine oxide broth on the detection of salmonellas using the Bactometer

Ninety-five salmonellas and 40 non-salmonellas were screened in the Bactometer using the standard formulation for Easter and Gibson's selenite cystine trimethylamine oxide dulcitol broth and versions in which dulcitol was replaced by mannitol or deoxyribose. More strains of salmonellas exceeded the current detection criteria (magnitude 250, rate 25) when dulcitol was replaced by either mannitol or deoxyribose as carbohydrate source. Using mannitol, more non-salmonella strains exceeded the detection criteria than with either dulcitol or deoxyribose.     

Sensitivity of Listeria monocytogenes to irradiation on poultry meat and in phosphate-buffered saline

The sensitivity of four strains of Listeria monocytogenes to irradiation on poultry meat and in phosphate-buffered saline was investigated. The D₁₀ values on poultry meat were 0.417–0.553 kGy depending on strain and plating medium used. Lower values were obtained in phosphate-buffered saline. Generally tryptone soya yeast extract and McBride agars gave a better recovery (higher D₁₀ value) than listeria selective agar. The D₁₀ values for L. monocytogenes were similar to those reported for Salmonella spp. irradiated under similar conditions. Therefore irradiation doses suggested to eliminate salmonellas from poultry carcasses would also be sufficient to remove L. monocytogenes.     

Fate of salmonellas and competing flora in meat sample enrichments in buffered peptone water and in Muller-Kauffmann's tetrathionate medium

Studies have been carried out in which growth patterns of a Salmonella sp. and competing micro-organisms, especially other Enterobacteriaceae, were followed during pre-enrichment in buffered peptone water (BPw) and subsequent selective enrichment in tetrathionate broth (TBB). Pre-enrichment cultures were inoculated with minced meat and three reference samples containing nalidixic acid-resistant salmonellas. Irrespective of their initial numbers in BPw, Enterobacteriaceae increased to 10⁸/ml or more. During incubation in TBB at 43C, numbers of lactose-positive Enterobacteriaceae decreased in most enrichments which resulted in a positive salmonella isolation, but remained constant in the majority of those that did not. Levels of lactose-negative Enterobacteriaceae did not decrease in most salmonella-positive tests, but did so in half of the salmonella-negative ones. In the salmonella-positive tests the numbers of salmonellas had increased to 10³–10⁷/ml in BPw and after transfer to TBB slowly reached 10⁴/ml or more. In all cases the numbers of salmonellas exceeded those of the competing flora on brilliant green agar (BGA). In the salmonella-negative tests the numbers of salmonellas had increased less in BPw and decreased in most of the TBB enrichments. In none of these negative tests did the numbers of salmonellas exceed those of the competing flora on BGA. Escherichia coli dominated in most of the salmonella-negative tests. The results suggest more influence of lactose-positive than lactose-negative Enterobacteriaceae on the detection of salmonellas. The effect of competing microorganisms seems to depend not only upon their initial numbers, but also upon the types that can interact with salmonellas during selective enrichment.     

Microbiological composition of raw milk from selected farms in the Camembert region of Normandy

Raw milk from 27 farms was sampled over 6 months for listerias, salmonellas, Yersinia enterocolitica and campylobacters. Total bacterial counts and somatic cell counts were measured. Lactococci, lactobacilli, dextran-producing leuconostocs, Brevibacterium linens , yeasts and moulds, Staphylococcus aureus and other Micrococcaceae, Pseudomonas , coliforms, Escherichia coli , enterococci, Clostridium perfringens and spores of anaerobic lactate-fermenting bacteria were also counted. Pseudomonas (2000 cfu ml⁻¹), lactococci (760 cfu ml⁻¹) and Micrococcaceae (720 cfu ml⁻¹) were the most numerous groups. Lactic acid bacteria were detected in all samples. Coliforms were present in most samples, but 84% of samples had counts <100 cfu ml⁻¹. Staphylococcus aureus was detected in 62% of milks, the average count was 410 cfu ml⁻¹. About 80% of supplies had ≤10 E. coli cfu ml⁻¹ and all samples had 1 Cl. perfringens cfu ml⁻¹. Two of the tested milks were positive for salmonellas (2·9%), four were positive for Listeria monocytogenes (5·8%), 25 for Yersinia enterocolitica (36%) and one for campylobacters (1·4%).