Isolation and initial characterization of the tellurite reducing moderately halophilic bacterium, Salinicoccus sp. strain QW6

Among the 49 strains of moderately halophilic bacteria isolated from the salty environments of Iran, a Gram-positive coccus designated as strain QW6 showed high capacity in the removal of toxic oxyanions of tellurium in a wide range of culture medium factors including pH (5.5-10.5), temperature (25-45 degrees C), various salts including NaCl, KCl, and Na(2)SO(4) (0.5-4M), selenooxyanions (2-10mM), and at different concentrations of potassium tellurite (0.5-1mM) under aerobic condition. Phenotypic characterization and phylogenetic analyses based on 16S rDNA sequence comparisons indicated that this strain was a member of the genus Salinicoccus. The maximum tellurite removal was exhibited in 1.5M NaCl at 35 degrees C, while the activity reduced by 53% and 47% at 25 and 45 degrees C, respectively. The optimum pH for removal activity was shown to be 7.5, with 90% and 83% reduced removal capacities at the two extreme values of 5.5 and 10, respectively. The impact of different concentrations of selenooxyanions (2-10mM) on tellurite removal by strain QW6 was evaluated. The ability of strain QW6 in the removal of tellurite in the presence of 6mM selenite increased by 25%. The concentration of toxic potassium tellurite in the supernatant of the bacterial culture medium decreased by 99% (from 0.5 to 0.005mM) after 6 days and the color of the medium changed to black due to the formation of less toxic elemental tellurium.     

Production and biological activity of laidlomycin, anti-MRSA/VRE antibiotic from Streptomyces sp. CS684

Culture broth of a streptomycete isolate, Streptomyces sp. CS684 showed antibacterial activity on methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE). Among purified substances from the organism, CSU-1, which is active against MRSA and VRE, is a C37H62O12Na (M+, 721.3875), and identified as laidlomycin. The anti-MRSA and anti-VRE activity of CSU-1 was stronger than oxacillin and vancomycin. Phylogenetic analysis showed that strain CS684 is very similar to Streptomyces ardus NRRL 2817T, whereas the ability of Streptomyces sp. CS684 to produce laidlomycin was shown to be unique.     

Evaluation of Media for Selective Isolation of Yeasts from Oral, Rectal, and Burn Wound Specimens

Six media were evaluated to determine their ability to isolate yeasts and inhibit bacteria. The media included the following: Snyder, Snyder tellurite, Sabouraud tellurite, Littman-gentamicin, molybdate, and Mycosel (BBL). Doses of mixed intestinal gram-negative bacilli and enterococci were most effectively inhibited by Snyder tellurite agar. Klebsiella pneumoniae was the most common bacterial contaminant of the other media. All six media were comparable in isolating yeasts while preventing the growth of the oral bacterial flora. The selection of a basal fungal growth medium for tellurite incorporation to inhibit bacteria but permit growth of yeasts was affected by pH. The bacteriostatic effect of tellurite was decreased with increasing pH of media while fungistatic action was increased. The arbitrary selection of Snyder and Littman agars to isolate yeast from burn wound cultures demonstrated the need to include a selective medium for these specimens. Blood, phenylethyl alcohol blood agar, and Columbia CN blood agar were all inadequate for isolating yeasts from burns. Growth of a variety of filamentous saprophytic and pathogenic dimorphic fungi grew adequately on four of five selective media tested.     

Selective enrichment with a resuscitation step for isolation of freeze-injured Escherichia coli O157:H7 from foods

We studied injury of Escherichia coli O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured E. coli O157:H7 cells from foods. Food samples inoculated with E. coli O157:H7 were stored for 16 weeks at -20 degrees C in a freezer. Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Large populations of E. coli O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples. In an experiment to detect E. coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modified E. coli broth without bile salts at 25 degrees C for 2 h and then selectively enriched at 42 degrees C for 18 h by adding bile salts and novobiocin. Our enrichment method was further evaluated by isolating E. coli O157:H7 from frozen foods inoculated with the organism prior to freezing. Two hours of resuscitation at 25 degrees C in nonselective broth improved recovery of E. coli O157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating E. coli O157:H7 from frozen foods contaminated with injured E. coli O157:H7 cells.     

费氏链霉菌中子辐射诱变菌株对新霉素的生物转化

对新霉素产生菌费氏链霉菌进行中子辐射诱变和突变株筛选, 获得不产新霉素的突变株。以突变株为转化菌种, 以新霉素为底物, 对转化发酵液进行高效液相色谱分析, 研究了不同转化条件对新霉素转化的影响。结果表明, 底物浓度、底物添加时间、底物添加方式、接种量、培养基装量、转化时间、碳源、氮源、pH、温度对新霉素转化具有不同程度的影响。以转化条件优化参数进行转化大量培养, 转化液经4步离子交换层析进行分离纯化, 薄层层析检测纯化样品为单一斑点。采用薄层生物自显影对获得的4个转化产物分离样品做生物活性检测, 发现4个样品对金黄色葡萄球菌和姜青枯假单孢杆菌都具有抑制活性, 1个样品对大白菜软腐样品具有明显的抑制活性。     

选择性富集沙门氏菌、副溶血弧菌和霍乱弧菌的共增菌培养基SVV

本研究通过单因素试验和响应面分析试验建立了能够选择性富集沙门氏菌、副溶血弧菌和霍乱弧菌的共增菌培养基SVV,采用平板计数法及三重荧光PCR技术验证了SVV的增菌效果。结果表明:SVV能同时富集以不同浓度比例混合的3种目标菌,37oC振荡培养18h后,菌体浓度达到105~108CFU/mL;SVV强烈抑制大部分的非目标菌;用荧光PCR方法检测经过37oC振荡培养18h的10份人工接种样品和608份实际样品,结果表明目标菌在SVV中增殖18h后菌量达到检测限以上,SVV联合荧光PCR检测方法的检出率为4.06%,比传统检测方法(3.78%)高,无假阴性和假阳性。SVV可望应用于水产品中沙门氏菌、副溶血弧菌和霍乱弧菌检测前的增菌处理,可简化检测过程,有效克服漏检,提高检出率。     

The sensitivity of Escherichia coli O157 to some antimicrobials by conventional and conductance assays

A range of sensitivities exhibited by Escherichia coli O157 to cefixime and potassium tellurite are demonstrated. The sensitivity was shown by growth on cefixime tellurite sorbitol MacConkey agar and by the effect on the metabolic activity in glucuronate trimethylamine-oxide conductance broth. These antimicrobials are regularly used in the isolation of this pathogen from food and clinical specimens, and such sensitivity may lead to the reporting of false negative samples.     

Description of Bacillus naganoensis sp. nov

A new species, Bacillus naganoensis, is proposed for an obligately aerobic, moderately acidophilic, endospore-forming bacterium that produces a thermostable, aciduric pullulanase (EC 3.2.1.41). The organism was isolated from soil by selection on solid, pullulan-containing medium at pH 4.0 and 30 degrees C. The isolate required a medium pH of less than 6.5 for growth initiation. Fatty acid composition studies revealed that the major fatty acid of cells grown in nutrient broth supplemented with 1% starch was 14-methylpentadecanoic acid (iso-C16) at 45 mol%. The guanine-plus-cytosine content of the DNA of this organism was 45 +/- 2 mol%. A type culture has been deposited with the American Type Culture Collection, Rockville, Md., as strain ATCC 53909.     

Comparison of various culture methods (Skirrow medium, a blood-free medium and a filtration system enriched in Bolton and Preston broths) for isolation of Campylobacter spp. from raw meat samples

We compared six procedures and investigated the optimal method for isolation of Campylobacter spp. from raw meat samples. Ninety-nine meat samples were enriched in Bolton broth and Preston broth, followed by plating on Skirrow, mCCDA, and blood agar (a membrane filter on its surface) media, respectively. Thirty-nine of 99 samples were positive and 71 Campylobacter were isolated by one or more methods. More than one species of Campylobacter were obtained in 8 (20.5 %) of 39 positive samples and two genotypes were yielded on the same medium (11 samples, 28.2 %) by pulsed-field gel electrophoresis (PFGE) genotyping. Enrichment by Preston broth was significantly better than by Bolton broth (P?<?0.05). Moreover, the latter failed to detect Campylobacter jejuni strains. Skirrow medium was significantly less efficient than mCCDA medium and membrane filtration method (P?<?0.05). Overall, the combination of PC (primary enrichment in Preston broth, followed by selective enrichment on mCCDA agar), PF (primary enrichment in Preston broth, followed by membrane filtration culture onto blood agar), and BF (primary enrichment in Bolton broth, followed by membrane filtration culture onto blood agar) methods provided the optimum isolation rate of Campylobacter spp.     

Methodology for recovery of chemically treated Staphylococcus aureus with neutralizing medium.

Recovery results of Staphylococcus aureus ATCC 6538 treated with phenolics and quaternary ammonium compounds on Dey and Engley (D/E) neutralizing medium at various time intervals were compared by the use of two commonly used media. Two recovery processes were utilized. In one, the chemically treated organisms were plated directly onto an agar medium. In the other, the aliquot was first put in broth and then was plated with agar. By either process, the numbers and the time period for recovery of organism were greater on D/E medium.     

Methodology for recovery of chemically treated Staphylococcus aureus with neutralizing medium

Recovery results of Staphylococcus aureus ATCC 6538 treated with phenolics and quaternary ammonium compounds on Dey and Engley (D/E) neutralizing medium at various time intervals were compared by the use of two commonly used media. Two recovery processes were utilized. In one, the chemically treated organisms were plated directly onto an agar medium. In the other, the aliquot was first put in broth and then was plated with agar. By either process, the numbers and the time period for recovery of organism were greater on D/E medium.     

人体肠道细菌寡营养培养组条件的优化研究

【目的】探讨寡营养对人体肠道细菌培养组的条件。【方法】通过稀释富集培养基、固体平板和增菌肉汤培养基成分获得寡营养培养基。对健康人粪便样本分别用原液(0)、5、10、20、30和40倍稀释的富集培养基(添加羊血和瘤胃液的血培养瓶)连续增菌,在不同时间点(第0、3、6、9、15、27、30天)吸取增菌液,用YCFA (yeast casitone fatty acid)固体培养平板分离菌落;用YCFA增菌肉汤增菌后再次挑取单菌落,利用基质辅助激光解吸/电离飞行时间(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF)质谱和16S rRNA基因测序鉴定菌株。通过比较上述6种寡营养条件分离肠道菌群的效果,选取富集培养基原液、稀释10倍和30倍这3 种条件下分离效果较好的富集条件,与同样稀释倍数条件的固体平板和增菌肉汤分别组合成9种培养基条件,进一步优化肠道菌群的培养组条件。【结果】在6种寡营养富集培养基中,未稀释(原液)、10 倍和30倍稀释的富集培养基分离细菌的种类比其他...     

Modification of sorbitol MacConkey medium containing cefixime and tellurite for isolation of Escherichia coli O157:H7 from radish sprouts

A modified version of sorbitol MacConkey medium containing cefixime and tellurite (CT-SMAC medium) was produced by adding salicin and 4-methylumbelliferyl-beta-D-galactopyranoside to CT-SMAC medium; this medium was designated CT-SSMAC medium and was used to isolate Escherichia coli O157:H7 from radish sprouts. Of 101 non-E. coli bacteria isolated from radish sprouts that produced colorless colonies similar to colonies of E. coli O157:H7 grown on CT-SMAC medium, 92 (91%) formed colonies that were red to pink or were beta-galactosidase negative and colorless on CT-SSMAC medium. On the other hand, colonies of E. coli O157:H7 strains were colorless and beta-galactosidase positive on CT-SSMAC medium. Our results suggest that CT-SSMAC medium is more selective than CT-SMAC medium for isolating E. coli O157:H7.     

Studies on Alternaria sesami pathogenic to sesame

Influence of pH on the growth of Alternaria sesami, its nutritional requirements and its ability to produce phytotoxic and antibacterial metabolites were tested. The isolate was cultured on Czapek-Dox broth and the culture filtrates were screened for phytotoxicity against seeds and seedlings of sesame. Chloroform extracts of the fungus exhibited antibacterial activity. Analysis of the culture filtrates for identifying toxins using chromatographic techniques revealed the presence of tenuazonic acid.     

Plaque formation by Tyzzer's organism in primary monolayer culture of mouse hepatocytes

Tyzzer's disease organism propagated on primary monolayer cultures of mouse hepatocytes and produced definite plaques. In phase contrast microscopy, the organisms were motile in the plaques. Plaque formation was inhibited by antiserum. After serial plaque cloning the organisms still had virulence in mice. To establish a standard plaquing procedure, factors affecting plaque formation were studied. The critical factors in plaque formation were the culture period of host cells before inoculation, medium for suspending the organisms, and temperature and time of infection. A 24 to 36 hr-preculture of host cells and trypticase soy broth (BBL) as the suspending medium gave the best results. The optimal conditions for infection were 37 C for 90 min. The plaquing efficiency was higher when a larger volume of inoculum was applied to host cell monolayers, suggesting that the organism played an active role in the initial stage of infection.     

The Microaerophile SPirillum volutans: Cultivation on Complex Liquid and Solid Media

Spirillum volutans grows only under microaerobic conditions in a peptone-succinate-salts broth, but can grow aerobically when the peptone is replaced by vitamin-free acid-hydrolyzed casein broth. The addition of potassium metabisulfite, norepinephrine, catalase or superoxide dismutase (SOD) permitted aerobic growth in peptone-succinate-salts broth. A combination of catalase and SOD had a synergistic effect. S. volutans lacked catalase and had only a low level of peroxidase activity, but did possess SOD activity (12 to 14 U/mg of protein). The organism was found to be extraordinarily sensitive to exogenous hydrogen peroxide. Illumination of peptone-succinate-salts broth generated hydrogen peroxide and rendered the medium inhibitory to growth. A combination of catalase and SOD prevented this inhibition. Growth of S. volutans on solid media, not previously possible, was accomplished by the use of vitamin-free acid-hydrolyzed casein and peptone-succinate-salts agar media; maximum growth responses were dependent on the following combination of factors: addition of bisulfite, catalase, or SOD, protection of the media from illumination, incubation in a highly humid atmosphere, and incubation under atmospheres of 12% oxygen or less. The results indicate that the microaerophilic nature of S. volutans is attributable largely to the high sensitivity of the organism to exogenous hydrogen peroxide and, to a lesser extent, superoxide radicals occurring in the culture medium.     

Shiga-toxigenic Escherichia coli O157 and non-Shiga-toxigenic E. coli O157 respond differently to culture and isolation from naturally contaminated bovine faeces

AIM: To quantify the effect of enrichment, immunomagnetic separation (IMS), and selective plating procedures on isolation of Shiga-toxigenic Escherichia coli O157 (STEC O157) and non-Shiga-toxigenic Escherichia coli O157 (non-STEC O157) from naturally contaminated bovine faeces. METHODS AND RESULTS: Two broth enrichment times, two IMS strategies, and two selective plating media were evaluated. STEC O157 and non-STEC O157 strains were often isolated from the same faecal specimen and responded differently to the isolation protocols. A large-volume IMS system was more sensitive than a conventional small-volume IMS method, but was also more expensive. STEC O157 was more frequently isolated from 6 h enriched broth and ChromAgar plates containing 0.63 mg l(-1) potassium tellurite (TCA). Non-STEC O157 was more frequently isolated from un-enriched broth and ChromAgar plates without tellurite (CA). CONCLUSIONS: The combination of 6-h enrichment in Gram-negative broth containing vancomycin, cefixime and cefsuludin, large volume IMS and selective plating on TCA maximized STEC O157 recovery from naturally contaminated cattle faecal specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: The pairing of proper enrichment with a specific plating procedure is key for STEC O157 recovery from naturally contaminated bovine faeces. Incorporating tellurite into an E. coli O157 detection strategy may select for the subset of E. coli O157 that contains the Shiga-toxin genes.     

Susceptibility tests for sulfamethoxazole-trimethoprim by a broth microdilution procedure

Most media used for susceptibility testing contain sulfonamide inhibitors that make them unacceptable for testing sulfonamides. The major substance that inhibits sulfonamides has been identified as thymidine, and recent efforts to remove it from Mueller-Hinton medium have made it possible to perform susceptibility tests with sulfamethoxazole-trimethoprim by broth microdilution. Some lots of Mueller-Hinton broth are thymidine free, but some contain small amounts of thymidine and require the addition of thymidine phosphorylase or lysed horse blood which contains thymidine phosphorylase. Some lots may contain too much thymidine so that its activity cannot be reversed by adding the recommended amont of thymidine phosphorylase. Therefore the suitability of a medium must be determined by testing with control strains. Some organism, for example enterococci, cannot be tested in thymidine phosphorylase-treated media because thymine works in the same way thymidine does to inhibit the action of sulfamethoxazole-trimethoprim.     

In vivo andin vitro cloning and phenotype characterization of tellurite resistance determinant conferred by plasmid pTE53 of a clinical isolate ofEscherichia coli

A determinant encoding resistance against potassium tellurite (Te(r)) was discovered in a clinical isolate of Escherichia coli strain KL53. The strain formed typical black colonies on solid LB medium with tellurite. The determinant was located on a large conjugative plasmid designated pTE53. Electron-dense particles were observed in cells harboring pTE53 by electron microscopy. X-Ray identification analysis identified these deposits as elemental tellurium and X-ray diffraction analysis showed patterns typical of crystalline structures. Comparison with JCPDS 4-0554 (Joint Committee on Powder Diffraction Standards) reference data confirmed that these crystals were pure tellurium crystals. In common with other characterized Te(r) determinants, accumulation studies with radioactively labeled tellurite showed that reduced uptake of tellurite did not contribute to the resistance mechanism. Tellurite accumulation rates for E. coli strain AB1157 harboring pTE53 were twice higher than for the plasmid-free host strain. In addition, no efflux mechanism was detected. The potassium tellurite resistance determinant of plasmid pTE53 was cloned using both in vitro and in vivo techniques in low-copy-number vectors pACYC184 and mini-Mu derivative pPR46. Cloning of the functional Te(r) determinant into high-copy cloning vectors pTZ19R and mini-Mu derivatives pBEf and pJT2 was not successful. During in vivo cloning experiments, clones with unusual "white colony" phenotypes were found on solid LB with tellurite. All these clones were Mucts62 lysogens. Their tellurite resistance levels were in the same order as the wild type strains. Clones with the "white" phenotype had a 3.6 times lower content of tellurium than the tellurite-reducing strain. Transformation of a "white" mutant with a recombinant pACYC184 based Te(r) plasmid did not change the phenotype. However, when one clone was cured from Mucts62 the "white" phenotype reverted to the wild-type "black" phenotype. It was suggested that the "white" phenotype was the result of an insertional inactivation of an unknown chromosomal gene by Mucts62, which reduced the tellurite uptake.     

Isolation, Identification, and Characterization of a Feather-Degrading Bacterium

A feather-degrading culture was enriched with isolates from a poultry waste digestor and adapted to grow with feathers as its primary source of carbon, sulfur, and energy. Subsequently, a feather-hydrolytic, endospore-forming, motile, rod-shaped bacterium was isolated from the feather-degrading culture. The organism was Gram stain variable and catalase positive and demonstrated facultative growth at thermophilic temperatures. The optimum rate of growth in nutrient broth occurred at 45 to 50°C and at pH 7.5. Electron microscopy of the isolate showed internal crystals. The microorganism was identified as Bacillus licheniformis PWD-1. Growth on hammer-milled-feather medium of various substrate concentrations was determined by plate colony count. Maximum growth (approximately 10⁹ cells per ml) at 50°C occurred 5 days postinoculation on 1% feather substrate. Feather hydrolysis was evidenced as free amino acids produced in the medium. The most efficient conditions for feather fermentation occurred during the incubation of 1 part feathers to 2 parts B. licheniformis PWD-1 culture (10⁷ cells per ml) for 6 days at 50°C. These data indicate a potential biotechnique for degradation and utilization of feather keratin.