Development of a Selective Medium for Isolation of Helicobacter pylori from Cattle and Beef Samples

Helicobacter pylori has been isolated from the human stomach with media containing only minimal selective agents. However, current research on the transmission and sources of infection requires more selective media due to the higher numbers of contaminants in environmental, oral, and fecal samples. The objective of this study was to develop and evaluate detection techniques that are sufficiently selective to isolate H. pylori from potential animal and food sources. Since H. pylori survives in the acidic environment of the stomach, low pH with added urea was studied as a potential selective combination. H. pylori grew fairly well on H. pylori Special Peptone plating medium supplemented with 10 mM urea at pH 4.5, but this pH did not sufficiently inhibit the growth of contaminants. Various antibiotic combinations were then compared, and a combination consisting of 10 mg of vancomycin per liter, 5 mg of amphotericin B per liter, 10 mg of cefsulodin per liter, 62,000 IU of polymyxin B sulfate per liter, 40 mg of trimethoprim per liter, and 20 mg of sulfamethoxazole per liter proved to be highly selective but still allowed robust colonies of H. pylori to grow. This medium was highly selective for recovering H. pylori from cattle and beef samples, and it is possible that it could be used to enhance the recovery of this bacterium from human and environmental samples, which may be contaminated with large numbers of competing microorganisms.     

一株新的溶磷草生欧文氏菌的分离、鉴定及其溶磷效果的初步研究

从湖南、湖北、云南等地磷矿开采场的土壤样品中筛选到一株溶磷能力较强的菌株P21,结合生理生化指标和16S rDNA序列分析鉴定其属于草生欧文氏菌菠萝变种(Erwinia herbicola var.ananas).该菌能溶解磷酸三钙、羟基磷灰石、磷酸铁、磷酸锌,其中对磷酸三钙和羟基磷灰石的每升液体培养基溶磷量(P2O5)分别高达1206.20mg、529.67mg.溶磷菌草生欧文氏菌菠萝变种P21对产地不同的8种磷矿石溶解能力不同,对云南晋宁和昆阳、四川雅安、江苏锦屏等地磷矿石有较强的溶解能力,每升液体培养基溶磷量分别为96.64mg、78.46mg、67.07mg、65.24mg,对其它产地的磷矿石溶解能力较差.实验表明,培养液的pH下降与溶磷菌P21的溶磷量无直接关系.     

Comparison of Media for the Isolation of Cryptococcus neoformans

Four media, Staib's Guizotia abyssinica, trypan blue, and Staib's with 2 and 10 mg of methyl violet per liter, were compared for the selective and differential isolation of Cryptococcus neoformans from environmental samples. Trypan blue medium allowed for the differentiation of C. neoformans colonies from Candida albicans colonies several days earlier than did Staib's medium. The addition of methyl violet to Staib's medium was found to be inhibitory to some strains of all species tested. Diphenyl in Staib's medium inhibited the growth of 30 strains of C. neoformans and C. albicans.     

Ampicillin-dextrin agar medium for the enumeration of Aeromonas species in water by membrane filtration

Published selective media were evaluated for the isolation of Aeromonas spp. from environmental samples by membrane filtration. Satisfactory recoveries were obtained only with mA agar (Rippey & Cabelli) and dextrin-fuchsin-sulphite agar (Schubert), but neither was sufficiently selective. The positive aspects of these two media were combined in a new medium, ampicillin-dextrin agar. Recovery from pure cultures and environmental samples was optimal at an ampicillin concentration of 10 mg/l and incubation for 24 h at 30 degrees C under aerobic conditions, and specificity was high (i.e. confirmation rate usually greater than 90%, no false negative colonies encountered). The medium can also be used for isolation of Aeromonas spp. from sea water provided that the vibriostatic agent 0/129 is added at 50 mg/l.     

Ampicillin-dextrin agar medium for the enumeration of Aeromonas species in water by membrane filtration

Published selective media were evaluated for the isolation of Aeromonas spp. from environmental samples by membrane filtration. Satisfactory recoveries were obtained only with mA agar (Rippey & Cabelli) and dextrin-fuchsin-sulphite agar (Schubert), but neither was sufficiently selective. The positive aspects of these two media were combined in a new medium, ampicillin-dextrin agar. Recovery from pure cultures and environmental samples was optimal at an ampicillin concentration of 10 mg/l and incubation for 24 h at 30°C under aerobic conditions, and specificity was high (i.e. confirmation rate usually <90%, no false negative colonies encountered). The medium can also be used for isolation of Aeromonas spp. from sea water provided that the vibriostatic agent 0/129 is added at 50 mg/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.     

Evaluation of Culture Techniques for Isolation of Pseudomonas pseudomallei from Soil

Three selective enrichment broths and four selective agar media were evaluated for their ability to support the growth of Pseudomonas pseudomallei both at 35°C and at ambient temperature (range, 20 to 32°C; mean, 25°C). Colony counts of 50 strains of P. pseudomallei and recovery studies with 1 soil strain in 60 simulated soil samples demonstrated that enrichment with Trypticase soy broth incorporating 5 mg of crystal violet per liter and 20 mg of colistin per liter (CVCB) and subculture to Ashdown medium supported the growth of all 50 strains and produced the highest recovery rates with the greatest suppression of other soil flora. An enrichment broth of MacConkey broth (purple) incorporating 10 mg of crystal violet per liter, 5 mg of bromcresol purple per liter, 25 mg of gentamicin per liter, and 650 mg of streptomycin per liter showed greater suppression of soil bacteria than CVCB, but it failed to support the growth of three strains of P. pseudomallei. Recovery rates were essentially the same irrespective of whether the soil samples were incubated at 35°C or at ambient temperature, provided cultures were incubated in protected shade for an extended period. This is an important feature for field work in large-scale epidemiological surveys in which resources are limited.     

Effect of the carbonic anhydrase inhibitor, acetazolamide, on Helicobacter pylori infection in vivo: a pilot study

BACKGROUND: Carbonic anhydrase inhibitors have been successfully used to treat peptic ulcers. Although carbonic anhydrase restriction does not inhibit Helicobacter pylori in vitro, recent studies suggest that carbonic anhydrase inhibition reduces the ability of H. pylori to survive in an acid environment as present in the stomach. METHODS: In a pilot study, we examined the effect of acetazolamide 500 mg as twice a day for 4 days in volunteers with active H. pylori infection. Effectiveness was judged by changes in the results of the urea breath test. RESULTS: Eight H. pylori infected volunteers completed the test. No urea breath test reverted to negative and there was a trend for the urea breath test value to increase [e.g. delta over baseline (DOB) mean +/- SE increased from 50.9 +/- 13 at baseline to 64.9 +/- 13 at day 5] during treatment with acetazolamide. CONCLUSION: The potential effect of carbonic anhydrase inhibitors on acid secretion may prevent effect on H. pylori in vivo and/or the sites of infection at the surface of the stomach may have a pH higher for any postulated acid-dependent effect to have an effect clinically.     

Gentamicin-based medium for the isolation of group D streptococci and application of the medium to water analysis.

Gentamicin-thallous-carbonate (GTC) medium contained (per liter): 40.0 g of Trypticase soy agar, 5.0 g of KH(2)PO(4), 2.0 g of NaHCO(2), 1.0 g of glucose, 1.0 g of esculin, 0.5 g of thallous acetate (TA), 0.5 g of ferric citrate, 0.75 ml of Tween 80, and 2.5 mg of gentamicin sulfate. The NaHCO(3) (20 ml of a 10% solution that had been heated to boiling) was added after sterilization of the basal medium. The spread plate technique was used to compare GTC agar with Pfizer selective enterococcus, TA, and KF agars by using sewage as well as bovine and swine fecal samples. Significantly greater numbers of group D streptococci were recovered on GTC agar than on Pfizer selective enterococcus or KF agars, within and over all samples. Higher counts also were obtained on GTC than on TA agar, but the differences were not statistically significant. The percentage of false positives was about the same for all four media. Samples of riverwater also were plated on GTC, TA, and KF agars, and significantly higher recoveries were obtained with GTC agar. GTC agar was superior to the other media examined primarily because of increased recoveries of Streptococcus bovis and S. equinus; other advantages of GTC agar were large colony size and short (24-h) incubation period. The percentage of false positives from riverwater was 13% for GTC agar and 0% for TA and KF agars; therefore, confirmation would be necessary when GTC agar is used with some types of environmental samples.     

Modified agar medium for detecting environmental salmonellae by the most-probable-number method.

Salmonellae in the environment remain a potential source of disease. Low numbers of salmonellae have been detected and enumerated from environmental samples by most-probable-number methods which require careful colony selection from a plated agar medium. A modified xylose lysine brilliant green medium was prepared to control the loss of selectivity caused by heating the brilliant green component. Added agar reduced colony spreading. The medium contained 47 g of xylose lysine agar base per liter; the agar content was adjusted to 2%, autoclaved, cooled to 50 degrees C, and then amended just before pouring to include H2S indicator and 7 ppm (7 ml of 1:1,000 brilliant green per liter) of unheated brilliant green dye. H2S-positive salmonellae were easily detected from sewage sludge compost to the exclusion of most other gram-negative bacteria. As a result, fewer non-salmonellae were picked for further most-probable-number analysis, greatly reducing the work load associated with the most-probable-number method. Direct plating was possible for enumerating salmonellae in laboratory composts containing ca. 10(3) or more salmonellae.     

Development of an internal control for evaluation and standardization of a quantitative PCR assay for detection of Helicobacter pylori in drinking water

Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori-specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.     

Modified agar medium for detecting environmental salmonellae by the most-probable-number method

Salmonellae in the environment remain a potential source of disease. Low numbers of salmonellae have been detected and enumerated from environmental samples by most-probable-number methods which require careful colony selection from a plated agar medium. A modified xylose lysine brilliant green medium was prepared to control the loss of selectivity caused by heating the brilliant green component. Added agar reduced colony spreading. The medium contained 47 g of xylose lysine agar base per liter; the agar content was adjusted to 2%, autoclaved, cooled to 50 degrees C, and then amended just before pouring to include H2S indicator and 7 ppm (7 ml of 1:1,000 brilliant green per liter) of unheated brilliant green dye. H2S-positive salmonellae were easily detected from sewage sludge compost to the exclusion of most other gram-negative bacteria. As a result, fewer non-salmonellae were picked for further most-probable-number analysis, greatly reducing the work load associated with the most-probable-number method. Direct plating was possible for enumerating salmonellae in laboratory composts containing ca. 10(3) or more salmonellae.     

Gentamicin-Based Medium for the Isolation of Group D Streptococci and Application of the Medium to Water Analysis

Gentamicin-thallous-carbonate (GTC) medium contained (per liter): 40.0 g of Trypticase soy agar, 5.0 g of KH₂PO₄, 2.0 g of NaHCO₂, 1.0 g of glucose, 1.0 g of esculin, 0.5 g of thallous acetate (TA), 0.5 g of ferric citrate, 0.75 ml of Tween 80, and 2.5 mg of gentamicin sulfate. The NaHCO₃ (20 ml of a 10% solution that had been heated to boiling) was added after sterilization of the basal medium. The spread plate technique was used to compare GTC agar with Pfizer selective enterococcus, TA, and KF agars by using sewage as well as bovine and swine fecal samples. Significantly greater numbers of group D streptococci were recovered on GTC agar than on Pfizer selective enterococcus or KF agars, within and over all samples. Higher counts also were obtained on GTC than on TA agar, but the differences were not statistically significant. The percentage of false positives was about the same for all four media. Samples of riverwater also were plated on GTC, TA, and KF agars, and significantly higher recoveries were obtained with GTC agar. GTC agar was superior to the other media examined primarily because of increased recoveries of Streptococcus bovis and S. equinus; other advantages of GTC agar were large colony size and short (24-h) incubation period. The percentage of false positives from riverwater was 13% for GTC agar and 0% for TA and KF agars; therefore, confirmation would be necessary when GTC agar is used with some types of environmental samples.     

Urea as sole source of nitrogen for plant growth

Summary Spirodela oligorrhiza grown in sterile culture was able to use urea as sole source of nitrogen but only when the pH of the culture medium was below 4.3. Plants inoculated into urea media at pH 6.4 initially made little growth and became nitrogen-deficient in appearance and composition although they contained about 100 grams of urea per gram fresh weight of tissue. After a period the pH of the medium usually fell below 4.3 and growth commenced. Growth with other compounds, e.g. ammonium, nitrate or allantoin, as sources of nitrogen was not similarly affected by the pH of the culture medium.Urease activity could always be detected in the tissues of Spirodela oligorrhiza growing on urea. Plants with little or no urease activity soon developed significant activity when inoculated into urea media at pH 4.0. When the pH of the medium was higher there was no increase in urease activity and no growth ensued. Plants growing on urea possessed an activity of about 50 milliunits per gram fresh weight of tissue, but if the pH of the medium fell to 3.5 or lower, the activity present rose to 10 times this level.Urease activity also appeared, in the absence of supplied urea, as plants became increasingly nitrogen-deficient.     

Isolation and enumeration of Campylobacter jejuni from poultry products by a selective enrichment method

A direct selective enrichment procedure was developed for the isolation of Campylobacter jejuni from poultry products. The selective enrichment medium (ATB) consisted of (per liter) tryptose (20 g), yeast extract (2.5 g), sodium chloride (5 g), FBP supplement (ferrous sulfate [0.25 g], sodium metabisulfite [0.25 g], sodium pyruvate [0.25 g]), bicine (10 g), and agar (1 g). Hematin solution (6.25 ml; prepared by dissolving 0.032 g of bovine hemin in 10 ml of 0.15 N sodium hydroxide solution and autoclaving at 0.35 kg/cm2 for 30 min), rifampin (25 mg), cefsulodin (6.25 mg), and polymyxin B sulfate (20,000 IU) were added after the medium was sterilized. The pH was adjusted to 8.0. Samples were enriched in the above medium at 42 degrees C for 48 h under an atmosphere of 5% O2, 10% CO2, and 85% N2. Enrichment cultures were streaked on a plating medium composed of Brucella agar, hematin solution, FBP supplement, and the above antibiotics. Plates were incubated under the same conditions as above. Suspect colonies from the plates were confirmed to be C. jejuni by morphological examination, growth characteristics, and biochemical tests. The above method yielded 25 isolates of C. jejuni from 50 samples of retail cut-up chicken and chicken parts, whereas a more complex method involving filtration, centrifugation, selective enrichment under a flowing atmosphere, and membrane filtration yielded only 6 positives from the same samples. The new isolation procedure was particularly effective in isolating C. jejuni in the presence of large numbers of Pseudomonas aeruginosa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and enumeration of Campylobacter jejuni from poultry products by a selective enrichment method.

A direct selective enrichment procedure was developed for the isolation of Campylobacter jejuni from poultry products. The selective enrichment medium (ATB) consisted of (per liter) tryptose (20 g), yeast extract (2.5 g), sodium chloride (5 g), FBP supplement (ferrous sulfate [0.25 g], sodium metabisulfite [0.25 g], sodium pyruvate [0.25 g]), bicine (10 g), and agar (1 g). Hematin solution (6.25 ml; prepared by dissolving 0.032 g of bovine hemin in 10 ml of 0.15 N sodium hydroxide solution and autoclaving at 0.35 kg/cm2 for 30 min), rifampin (25 mg), cefsulodin (6.25 mg), and polymyxin B sulfate (20,000 IU) were added after the medium was sterilized. The pH was adjusted to 8.0. Samples were enriched in the above medium at 42 degrees C for 48 h under an atmosphere of 5% O2, 10% CO2, and 85% N2. Enrichment cultures were streaked on a plating medium composed of Brucella agar, hematin solution, FBP supplement, and the above antibiotics. Plates were incubated under the same conditions as above. Suspect colonies from the plates were confirmed to be C. jejuni by morphological examination, growth characteristics, and biochemical tests. The above method yielded 25 isolates of C. jejuni from 50 samples of retail cut-up chicken and chicken parts, whereas a more complex method involving filtration, centrifugation, selective enrichment under a flowing atmosphere, and membrane filtration yielded only 6 positives from the same samples. The new isolation procedure was particularly effective in isolating C. jejuni in the presence of large numbers of Pseudomonas aeruginosa.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Helicobacter pylori UreI protein: role in adaptation to acidity and identification of residues essential for its activity and for acid activation

Helicobacter pylori is a human gastric pathogen that survives the strong acidity of the stomach by virtue of its urease activity. This activity produces ammonia, which neutralizes the bacterial microenvironment. UreI, an inner membrane protein, is essential for resistance to low pH and for the gastric colonization of mice by H. pylori. In the heterologous Xenopus oocytes expression system, UreI behaves like an H+-gated urea channel, and His-123 was found to be important for low pH activation. We investigated the role of UreI directly in H. pylori and showed that, in the presence of urea, strains expressing wild-type UreI displayed very rapid stimulation of extracellular ammonia production upon exposure to pH 相似文献   

Isolation of mycobacteria from acidic forest soil samples: comparison of culture methods

To evaluate which combination of decontamination method and medium is most reliable when examining acidic, organic forest soils for mycobacteria, three decontamination methods and five media supplemented with cycloheximide were compared. Before decontamination, the samples were incubated at 37°C for 5 h to allow germination of microbial spores. The recovery of mycobacteria was significantly influenced both by the method and by medium. Decontamination with NaOH or H₂SO₄ both combined with malachite green and cycloheximide yielded higher viable counts of mycobacteria than decontamination with NaOH followed by oxalic acid. Egg media at pH 5·5 resulted in lower mycobacterial counts than egg media at pH 6·5 or Mycobacteria 7H11 agar. The numbers of slopes totally free of contaminants revealed Mycobacteria 7H11 agar medium to be more prone to contamination than the four egg media tested. The highest counts of mycobacteria and a low rate of contamination were obtained when decontamination with NaOH-malachite green–cycloheximide was combined with culture on glycerol and cycloheximide supplemented egg medium at pH 6·5.     

Trial of different nutrient media for the production of an intracellular thermolabile enterotoxin by Escherichia coli strains H74-114 and 86

The conditions of cultivation, ensuring the maximum accumulation of intracellular thermolabile enterotoxin in the cultures of two E. coli strains of different origin, have been studied. Culture media manufactured in the USSR have been selected and the conditions of cultivation, necessary for obtaining intracellular thermolabile enterotoxin in preparative amounts, have been established. Under these conditions the yield of thermolabile enterotoxin in 1.4 mg per 1 liter of culture medium for strain H74-114 and 1.0 mg per 1 liter of culture medium for strain 86.     

Factors Influencing the Effectiveness of Swimming Pool Bactericides

Techniques for culturing, harvesting, and testing bacteria to evaluate bactericidal chemicals for swimming pools are described. Concentrations of 25, 50, and 100 mg of the chlorine stabilizer cyanuric acid per liter increased the time required for a 99% kill of Streptococcus faecalis by 0.5 mg of chlorine per liter at pH 7.4 and 20 C from less than 0.25 min without cyanuric acid to 4, 6, and 12 min, respectively. The effect of concentrations of ammonia nitrogen in the range found in swimming pools on the rate of kill of 0.5 mg of chlorine per liter and of chlorine plus cyanuric acid was tested. At concentrations of ammonia nitrogen greater than 0.05 mg per liter, faster rates of kill of S. faecalis were obtained with 100 mg of cyanuric acid per liter plus 0.5 mg of chlorine per liter than with 0.5 mg of chlorine per liter alone. When water samples from four swimming pools with low ammonia levels were used as test media, 0.5 mg of added chlorine per liter killed 99.9% of the added S. faecalis in less than 2 min, but water from a pool with a large number of children required 60 to 180 min of treatment.