Cloning of the phs genetic locus from Salmonella typhimurium and a role for a phs product in its own induction.

The Salmonella typhimurium phs chromosomal locus essential for the reduction of thiosulfate to hydrogen sulfide was cloned, and some features of its regulation were examined. The phs locus conferred H2S production on Escherichia coli, suggesting that it contains the structural gene for thiosulfate reductase. H2S production by the E. coli host was, as in S. typhimurium, suppressed by nitrate or glucose in the growth medium. The presence of plasmid-borne phs genes in a S. typhimurium chl+ host containing a chromosomal phs::lacZ operon fusion was found to significantly increase the relative induction efficiency of beta-galactosidase by thiosulfate. These results are consistent with a model for phs regulation in which the true inducer is not thiosulfate per se and in which the action of a phs-encoded molybdoprotein, possibly the reductase itself, converts thiosulfate into a compound that resembles the true inducer more closely than does thiosulfate.     

Comparison of the Rates of Motility of Salmonella with Those of Other Enteric Bacteria

Comparison of the rates of motility through a semisolid medium of 16 common Salmonella sp., 14 Escherichia coli serotypes, 4 Arizona strains, 2 Escherichia freundii (Citrobacter) isolates, 2 Proteus sp., and 2 Pseudomonas sp. revealed the following. (i) Very closely related bacteria could demonstrate markedly different rates of progression. (ii) All of the salmonellae tested advanced faster than the Proteus and Pseudomonas test cultures but some Salmonella sp., notably S. choleraesuis and S. typhi, progressed relatively slowly compared to many other test cultures. (iii) The mean rate of motility for the fastest 14 Salmonella sp. (1.49 cm/hr) was not statistically greater than the mean value for the 14 E. coli serotypes (1.31 cm/hr) at the 1% level of significance. Selective motility procedures may not be a reliable means of isolating all Salmonella sp. from materials contaminated with other bacteria.     

Observations on brilliant green agar with H2S indicator.

Several formulations of brilliant green agar with an added H2S indicator were evaluated. Results were optimum with variations of a basic formula consisting of 40 g of tryptic soy agar (Difco), 8 g of lactose, 8 g of sucrose, 80 mg of phenol red, 1 g of sulfanilamide, 1.5 g of ferric ammonium citrate, 5 g of sodium thiosulfate pentahydrate, and 7 mg of brilliant green dye per liter. Brilliant green dye was added after sterilization of the other components This formulation supported good growth of all of 39 strains of Salmonella tested. Normal biochemical types formed pink colonies with black centers, and an H2S-negative S. choleraesuis formed pink colonies without black centers. Of other bacteria tested, only Enterobacter, Klebsiella, and a few Citrobacter strains showed significant growth in 24 h. When lactose was omitted from the formulation, a lactose-fermenting strain formed pink colonies with black centers, and differentiation of Salmonella from the Enterobacter-Klebsiella groups was equally good. Addition of xylose (4.0 g) and L-lysine hydrochloride (5.4 g) to the above formulation improved differentiation between Salmonella and the few Citrobacter strains that grew and produced more intense blackening in Salmonella colonies. Addition of an H2S indicator to brilliant green agar formulations aided in identification of Salmonella colonies, especially in mixtures with other bacteria. These media were judged to give better differentiation of salmonellae from other bacteria than Hektoen agar with added novobiocin (10 mg/liter).     

Observations on brilliant green agar with H2S indicator.

Several formulations of brilliant green agar with an added H2S indicator were evaluated. Results were optimum with variations of a basic formula consisting of 40 g of tryptic soy agar (Difco), 8 g of lactose, 8 g of sucrose, 80 mg of phenol red, 1 g of sulfanilamide, 1.5 g of ferric ammonium citrate, 5 g of sodium thiosulfate pentahydrate, and 7 mg of brilliant green dye per liter. Brilliant green dye was added after sterilization of the other components This formulation supported good growth of all of 39 strains of Salmonella tested. Normal biochemical types formed pink colonies with black centers, and an H2S-negative S. choleraesuis formed pink colonies without black centers. Of other bacteria tested, only Enterobacter, Klebsiella, and a few Citrobacter strains showed significant growth in 24 h. When lactose was omitted from the formulation, a lactose-fermenting strain formed pink colonies with black centers, and differentiation of Salmonella from the Enterobacter-Klebsiella groups was equally good. Addition of xylose (4.0 g) and L-lysine hydrochloride (5.4 g) to the above formulation improved differentiation between Salmonella and the few Citrobacter strains that grew and produced more intense blackening in Salmonella colonies. Addition of an H2S indicator to brilliant green agar formulations aided in identification of Salmonella colonies, especially in mixtures with other bacteria. These media were judged to give better differentiation of salmonellae from other bacteria than Hektoen agar with added novobiocin (10 mg/liter).     

Influence of pH of TSI medium on the detection of hydrogen sulfide production by Campylobacter hyointestinalis

AIMS: To examine the influence of pH of triple sugar iron (TSI) agar medium on the detection of hydrogen sulfide (H(2)S) production in Campylobacter hyointestinalis ssp. hyointestinalis (CHH). METHODS AND RESULTS: TSI medium was adjusted by the addition in HCl or NaOH to cover a pH 6.0-9.0. One loopful of bacterial growth of CHH strain ATCC 35217 was inoculated into each different pH medium, and incubated at 37 degrees C under micro-aerobic conditions. The H(2)S production was not detectable even after incubation for 72 h in acidic medium pH; however, TSI with alkaline pH (8.0-9.0) allowed detection as early as 3 h of incubation. A total of 20 CHH strains from various animal sources were examined for the detection of H(2)S production in TSI medium with pH 9.0. The H(2)S was detected in all the strains examined within 12 h, and the judgment was unambiguous. CONCLUSION: The results showed that the detection of H(2)S production by CHH was influenced by medium pH, and TSI with alkaline condition is highly sensitive. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the present study suggest that TSI medium with alkaline pH contributes to fast detection and led to unambiguous judgment of H(2)S production by CHH.     

R/B Enteric Differential System for Identification of Enterobacteriaceae

The R/B Enteric Differential System for identifying enteric bacteria has been evaluated with 451 "unknown" cultures from the stock culture collection of the Center for Disease Control. An average of 89.6% of these cultures were correctly identified by the R/B system, when used as recommended by the manufacturer but without the assistance of serology. This percentage ranged, however, from 47% for Klebsiella to 100% for Serratia and Providencia. Of 11 groups or genera of Enterobacteriaceae tested, only three (Enterobacter, Serratia, and Providencia) were identified with 95% or better accuracy. Four groups (Arizona, Citrobacter, Escherichia, and Salmonella) attained 90 to 95% accuracy of identification, and three groups (Edwardsiella, Proteus, and Shigella) scored between 85 and 90% accuracy. We recommend the R/B system as a screening device which is reasonably successful in grouping bacteria but not as a substitute for more exacting conventional procedures.     

Evaluation of a quantitative screening method for hydrogen sulfide production by cheese-ripening microorganisms: the first step towards l-cysteine catabolism

A practical adaptation of the methylene blue reaction for hydrogen sulfide quantification was developed to perform microbial selection. Closed plate flasks containing a zinc-agar layer above the liquid microbial culture are proposed as a trap system where the H(2)S can be retained and then quantified by the methylene blue reaction. Using this quantitative method, the ability to produce H(2)S was studied in several cheese-ripening microorganisms. Our aim was to select strains that produce the highest quantities of H(2)S as the main product of L-cysteine catabolism. Thirty seven yeast and bacteria strains were cultivated with or without L-cysteine. The separation between the growth medium and the H(2)S trapping layer displayed good performance: all the studied strains grew efficiently and only negligible loss of H(2)S was observed during culturing. The strains displayed large differences in their H(2)S production capabilities: yeast strains were greater producers of H(2)S than bacteria with production strain-related in both cases. Furthermore, the relationship between H(2)S production and L-cysteine consumption was analyzed, which made it possible for us to select microorganisms with high capacity in L-cysteine degradation. The production of volatile sulfur compounds was also studied and the possible effect of culture pH and metabolic differences between strains are discussed.     

Development of a method to measure hydrogen sulfide in wine fermentation

A hydrogen sulfide (H2S) detecting tube was developed for the quantitative determination of H2S produced by yeast during laboratory scale wine fermentations. The detecting tube consisted of a small transparent plastic tube packed with an H2S-sensitive color-indicating medium. The packed medium changed color, with the color change progressing upward from the bottom of the tube, upon exposure to H2S produced by yeast during fermentation. A calibration study using a standard H2S gas showed that the length of the portion that darkened was directly related to the quantity of H2S (microg) with a high correlation coefficient (r2=0.9997). The reproducibility of the H2S detecting tubes was determined with five repetitive measurements using a standard H2S solution [5.6 microg/200 ml (28 ppb)], which resulted in a coefficient of variation of 3.6% at this level of H2S. With the sulfide detecting tubes, the production of H2S was continuously monitored and quantified from laboratory scale wine fermentations with different yeast strains and with the addition of different levels of elemental sulfur to the grape juice. This sulfide detecting tube technology may allow winemakers to quantitatively measure H2S produced under different fermentation conditions, which will eventually lead winemakers to better understand the specific factors and conditions for the excessive production of H2S during wine fermentation in a large production scale.     

Development and evaluation of two novel oligonucleotide probes based on 16S rRNA sequence for the identification of Salmonella in foods

DNA sequence in the V3 to V6 region of the 16S rRNA gene of Salmonella enteritidis was determined. By comparison of this sequence with those of Escherichia coli and Proteus vulgaris obtained from GenBank/EMBL database, three oligonucleotides termed as 16S I, 16S II and 16S III were synthesized. Hybridization of these oligonucleotides with 325 Salmonella isolates and some non- Salmonella isolates including the Salmonella closely related species of the family of Enterobacteriaceae showed that 16S II could not be used as a Salmonella specific-probe. 16S I and 16S III hybridized with all the Salmonella isolates tested, the former also hybridizing with Citrobacter spp. and the latter hybridizing with Klebsiella pneumoniae as well as Serratia marcescens. Since enrichment of the target cells in food samples was usually required prior to the DNA hybridization assay, the interference from those non- Salmonella isolates could be prevented by enrichment by culturing in lactose-combined tetrathionate (CTET) broth followed by Gram-negative (GN) broth at 37°C and/or 43°C. Such a culture step could inhibit the growth of Klebsiella spp., Ser. marcescens and/or Citrobacter spp. and allowed the specific detection of Salmonella .     

The phs gene and hydrogen sulfide production by Salmonella typhimurium.

Salmonella typhimurium produces H2S from thiosulfate or sulfite. The respective pathways for the two reductions must be distinct as mutants carrying motations in phs, chlA, and menB reduced sulfite, but not thiosulfate, to H2S, and glucose repressed the production of H2S from thiosulfate while it stimulated its production from sulfite. The phs and chlA mutants also lacked a methyl viologen-linked thiosulfate reductase activity present in anaerobically grown wild-type cultures. A number of hydroxylamine, transposon Tn10 insertion, and Mu d1(Apr lac) operon fusion mutants defective in phs were characterized. One of the hydroxylamine mutants was an amber mutant, as indicated by suppression of its mutation in a supD background. The temperature-sensitive phs mutants produced H2S and methyl viologen-linked thiosulfate reductase at 30 degrees C but not at 42 degrees C. The reductases in all such mutants grown at 30 degrees C were as thermostable as the wild-type enzyme and did not differ in electrophoretic relative mobility, suggesting that phs is not the structural gene for thiosulfate reductase. Expression of beta-galactosidase in phs::Mu d1(Apr lac) mutants was dependent on anaerobiosis and the presence of reduced sulfur. It was also strongly influenced by carbon source and growth stage. The results are consistent with a model in which the phs gene encodes a regulatory protein essential for the reduction of thiosulfate to hydrogen sulfide.     

Enhanced contrast of bacteriophage plaques in Salmonella with ferric ammonium citrate and sodium thiosulfate (FACST) and tetrazolium red (TZR)

Visualization of bacteriophage plaques may be enhanced by addition of ferric ammonium citrate and sodium thiosulfate (FACST) or 2,3,5-triphenyltetrazolium chloride (tetrazolium red, TZR) to the soft agar layer of a traditional bacteriophage plaque assay. Background color from these reagents improved contrast between clear plaques and turbid host lawns in trypticase soy agar (TSA) plates. Enhancement by FACST is based on reaction with hydrogen sulfide gas (H2S) produced by some strains of bacteria and was tested here using H2S+ and H2S- strains of Salmonella enterica subsp. enterica with a bacteriophage (Podoviridae) isolated from swine lagoon effluent. Only the H2S+ strain produced dark brown-black color in FACST-amended agar. Both strains showed bright pinkish-red color in TZR-amended agar. Color intensity for both reagents decreased with decreasing concentrations of the reagents. Contrast in FACST-amended plates appeared greater than that with TZR, but diminished after 12 h, while contrast in TZR-amended plates remained constant. At the concentrations tested, neither reagent affected plaque counts in the H2S+ strain. The FACST should be useful in bacteriophage plaque assays with H2S+ strains of Salmonella and other H2S+ bacteria.     

A method for controlling hydrogen sulfide in water by adding solid phase oxygen

This work evaluates the addition of solid phase oxygen, a magnesium peroxide (MgO(2)) formulation manufactured by Regenesis (oxygen-releasing compounds, ORC), to inhibit the production of hydrogen sulfide (H(2)S) in an SRB-enriched environment. The initial rate of release of oxygen by the ORC was determined over a short period by adding sodium sulfite (Na(2)SO(3)), which was a novel approach developed for this study. The ability of ORCs to control H(2)S by releasing oxygen was evaluated in a bench-scale column containing cultured sulfate reducing bacteria (SRB). After a series of batch tests, 0.4% ORC was found to be able to inhibit the formation of H(2)S for more than 40 days. In comparison, the concentration of H(2)S dropped from 20 mg S/L to 0.05 mg S/L immediately after 0.1% hydrogen peroxide (H(2)O(2)) was added, but began to recover just four days later. Thus, H(2)O(2) does not seem to be able to inhibit the production of sulfide for an extended period of time. By providing long-term inhibition of the SRB population, ORC provides a good alternative means of controlling the production of H(2)S in water.     

Hydrogen sulfide production and fermentative gas production by Salmonella typhimurium require F0F1 ATP synthase activity.

A previously isolated mutant of Salmonella typhimurium lacking hydrogen sulfide production from both thiosulfate and sulfite was shown to have a single mutation which also caused the loss of fermentative gas production and the ability to grow on nonfermentable substrates and which mapped in the vicinity of the atp chromosomal locus. The implication that F0F1 ATP synthase might be essential for H2S and fermentative gas production was explored. The phs plasmid conferring H2S production on wild-type Escherichia coli failed to confer this ability on seven of eight E. coli atp point mutants representing, collectively, the eight genes encoding the subunits of F0F1 ATP synthase. However, it did confer some thiosulfate reductase activity on all except the mutant with a lesion in the ATP synthase catalytic subunit. Localized mutagenesis of the Salmonella atp chromosomal region yielded 500 point mutants unable to reduce thiosulfate to H2S or to produce gas from glucose, but differing in the extents of their ability to grow on succinate, to perform proton translocation as measured in a fluorescence quenching assay, and to reduce sulfite to H2S. Biochemical assays showed that all mutants were completely devoid of both methyl viologen and formate-linked thiosulfate reductase and that N,N'-dicyclohexylcarbodiimide blocked thiosulfate reductase activity by the wild type, suggesting that thiosulfate reductase activity has an absolute requirement for F0F1 ATP synthase. Hydrogenase-linked formate dehydrogenase was also affected, but not as severely as thiosulfate reductase. These results imply that in addition to linking oxidation with phosphorylation, F0F1 ATP synthase plays a key role in the proton movement accompanying certain anaerobic reductions and oxidations.     

Factors affecting iron sulfide-enhanced bacteriophage plaque assays in Salmonella

Reaction of ferric ions with hydrogen sulfide (H(2)S) enhances contrast of phage plaques in H(2)S+ Salmonella, but contrast diminishes in weak H(2)S+ strains. H(2)S was affected by concentrations of peptones, glucose, ferric ammonium citrate (FAC) and sodium thiosulfate (ST), and by FAC:ST ratio, temperature, pH, air, and host strain. Increasing peptone levels was most important for improving contrast in weak H(2)S+ strains.     

Stable isotope fractionation by Clostridium pasteurianum. 2. Regulation of sulfite reductases by sulfur amino acids and their influence on sulfur isotope fractionation during SO32- and SO42- reduction

In addition to an assimilatory sulfite reductase, studies of cultures of Clostridium pasteurianum supplemented with methionine, cysteine, and 35SO42- provides evidence for another reductase which is induced by SO32-. This inducible reductase appears to be dissimaltory because of the copious sulfide production arising when the cells are grown on SO32-. Cysteine can repress the assimilatory sulfite reductase but does not affect the inducible reductase. During late logarithmic growth on 1 mM SO42- + 10mM cysteine, depression of the inducible reductase occurred along with increased sulfide production. The presence of 1 mM cysteine and (or) 1 mM cysteine and (or) 1 mM methionine does not affect the inverse sulfur isotope effect for evolved H2S. However, 5 and 10 mM cysteine reduce the maximum delta34S value for released H2S from +40 to 10%. A small conversion of cysteine to H2S by C. pasteurianum occurs, but only in the stationary phase.     

Novel mechanism for conditional aerobic growth of the anaerobic bacterium Treponema denticola

Treponema denticola, a periodontal pathogen, has recently been shown to exhibit properties of a facultative anaerobic spirochete, in contrast to its previous recognition as an obligate anaerobic bacterium. In this study, the capacity and possible mechanism of T. denticola survival and growth under aerobic conditions were investigated. Factors detrimental to the growth of T. denticola ATCC 33405, such as oxygen concentration and hydrogen sulfide (H(2)S) levels as well as the enzyme activities of gamma-glutamyltransferase, cysteinylglycinase, and cystalysin associated with the cells were monitored. The results demonstrated that T. denticola grew only at deeper levels of broth (>or=3 ml in a 10-ml tube), high inoculation ratios (>or=20% of culture in medium), and short cultivation times (相似文献   

Heterotrophic sulfur reduction by Thermotoga sp. strain FjSS3.B1

Thermotoga sp. strain FjSS3.B1 was able to reduce sulfur to sulfide when grown on a mineral medium with glucose as the sole carbon and energy source. There was no increase in specific growth yield coupled to sulfur reduction, but the specific growth rate, final growth yield, and tolerance of H2 were all increased in the presence of sulfur. At dissolved H2 concentrations, of 550 to 600 mumol/l (at 77 degrees C) growth was not possible unless sulfur was added. Glucose was fermented via the Embden-Meyerhof-Parnas pathway to lactate, acetate, H2 and CO2 (and other unidentified minor products). The thermodynamic problems associated with the relatively high redox potential electrons from the 1,3-bisphosphoglycerate/glyceraldehyde 3-phosphate couple (E'0 = -350 mV) are overcome by reducing sulfur to sulfide (E'0 = -270 mV) rather than the energetically unfavourable production of H2 (E'0 = -414 mV). Under high hydrogen partial pressures there was increased production of lactate as an alternative electron sink. The results indicate that sulfur reduction operates primarily as an electron sink rather than as a detoxification reaction or energy-generating mechanism.     

Factors affecting salmonellae repopulation in composted sludges.

The repopulation potential and recovery of Salmonella sp. and their close relatives Arizona spp. and Citrobacter spp. in sewage sludge which had been composted was examined. Salmonellae growth in previously composted sludge was found to occur in the mesophilic temperature range (20 to 40 degrees c), require a moisture content of greater than or equal to 20%, and require a carbon/nitrogen ratio in excess of 15:1.     

Development of a plating medium for selection of Helicobacter pylori from water samples

The goal of this study was to develop a simple plating medium to allow large-scale screening of water samples for the presence of Helicobacter pylori. Five conventional plating media (brain heart infusion, brucella agar, Columbia blood agar base, campylobacter agar kit Skirrow, and HPSPA medium), each containing a commercial antibiotic supplement, were initially evaluated. Eight strains selected as common waterborne organisms (Acinetobacter, Aeromonas, Bacillus, Escherichia coli, Enterobacter, Enterococcus, Helicobacter pylori, and Pseudomonas strains) were individually plated onto each of these media. Three organisms (Acinetobacter, E. coli, and H. pylori) were able to grow on all five media. This growth was unacceptable since Helicobacter grows very slowly and competing organisms must be inhibited for up to 7 days. Therefore, a more selective medium (HP agar) containing a novel mixture of growth supplements plus amphotericin B and polymyxin B was developed. This medium also included a phenol red color indicator for urease production. Aliquots of nonsterile well water that contained native flora (Flavobacterium, Serratia, Citrobacter, Pasteurella, Ochrobactrum, Rahnella, and unidentified molds) and were further adulterated with the eight strains listed above (10(6) CFU of each strain per 100 ml) were spiked with H. pylori and were plated. In spite of the heavy mixed microbial load, only H. pylori colonies grew during 7 days of incubation at 37 degrees C. The color indicator system allowed presumptive identification of H. pylori colonies sooner (12 to 20 h) than the conventional media tested allowed. The HP formulation developed in this study provides a medium with superior selectivity for H. pylori from mixed microbial populations in water and reduces the time required to complete the assay.