Reagent Strips and Conventional Tests for Acid Production from Mannitol and Coagulase Activity of Staphylococcus: a Comparative Study

A total of 244 Staphylococcus strains were tested simultaneously for acid production from mannitol and for coagulase activity with reagent-impregnated paper strips and with their conventional counterparts. Significant correlation was obtained with 97.9% of the strains for mannitol and with 95% for the coagulase test. The paper strip method is a combined test for both mannitol and coagulase tests, thus making it more convenient and simpler than conventional methods. The results are obtained rapidly within 6 hr by the paper strip method. However, as the paper strip method is designed for the aerobic system, the conventional tests were also carried out under aerobic conditions to compare the results.     

Rapid tests for esculin hydrolysis by anaerobic bacteria

Esculin hydrolysis is one of the biochemical tests used in the identification of anaerobic microorganisms. The conventional method by use of growing microbial cells requires 24–48 hours of incubation. On the other hand, growth independent methods like the buffered esculin test, the spot test, and the PathoTec strip test utilize the presence of constitutive enzymes and, therefore, yield results in 1–4 hours. A total of 817 anaerobic organisms were used in this study to determine the sensitivity and specificity of the three rapid methods. All three rapid methods gave excellent correlation with the standard conventional method. Over 99% of the organisms gave comparable results with the spot test and the buffered esculin test within one hour; the PathoTec strip test required up to 4 hours. The former two were not only more rapid but also more economical than the PathoTec strip test.     

大蒜素抗变异链球菌的致龋效果

目的 研究大蒜素对口腔变异链球菌生长及其菌斑生物膜粘附的抑制作用。方法 二倍稀释法梯度稀释测最小抑菌浓度（minimum inhibitory concentration,MIC）,将MIC以上2个梯度浓度对应的培养物涂布于BHI培养基上进行次代培养获得最低杀菌浓度（minimum bactericidal concentration,MBC）;酶标仪测A值观察不同浓度大蒜素抑菌效应;抑制产酸试验观察抑制细菌产酸效应;结晶紫法研究亚抑菌浓度提取物对变异链球菌粘附能力及生物膜总量的影响;采用激光共聚焦荧光显微镜（laser scanning confocal microscopy,LSCM）观察常态牙菌斑生物膜生长过程中及药物处理后牙菌斑生物膜中死菌和活菌的构成,研究其对牙菌斑生物膜结构和活性的影响。结果 抑菌试验中,得到大蒜素MIC为12.8 mg/L,MBC为25.8 mg/L。MIC及亚抑菌浓度抑菌试验显示均有一定的抑菌性,抑制率为2.17%~67.12%,并且抑菌性与浓度梯度成正相关。产酸试验显示24 h内大蒜素明显抑制细菌产酸（P<0.01）,细菌粘附试验结果显示大蒜素在MIC时生物膜的生成速度最慢,生物膜的总量最低（P<0.01）。共聚焦荧光显微镜可见大蒜素组随药物浓度增加,菌斑生物膜较薄,绿色的活菌及团块明显减少,抑制生物膜的生长。结论 大蒜素对变异链球菌生长、产酸与粘附有一定抑制作用。     

Escherichia coli Tryptophanase in the Enteric Environment

The activity of the enzyme tryptophanase in the enteric environment was investigated to elucidate the significance of the enzyme in the metabolism of Escherichia coli. The tryptophanase activity, tryptophan content, and indole concentration as well as the numbers of E. coli were determined in the intestinal and fecal contents of conventional, germ-free, and monocontaminated axenic laboratory mice. Increasing the tryptophan content of the diet of mice having a conventional microflora increased the tryptophanase activity of the enteric microflora by a factor of almost 2 but did not increase the numbers of E. coli either absolutely or relative to other facultative enteric coliforms. In the enteric environment, E. coli is responsible for very little tryptophanase activity, a fraction calculated to be less than 0.02%. The values for the experimental parameters were much the same in the contents of the cecum and in the fecal material.     

Tryptophan Production by a Lipoic Acid Auxotroph of Enterobacter aerogenes Having Both Pyruvic Acid Productivity and High Tryptophanase Activity

A new process for tryptophan production was established using a lipoic acid auxotrophic mutant, Enterobacter aerogenes l-12, which has both pyruvic acid productivity and tryptophanase activity. The process consists of the production of pyruvic acid from glucose by the washed cells and the subsequent conversion of the acid to tryptophan by the tryptophanase itself in the presence of indole and NH₄C1.

To prepare washed cells of which the tryptophanase activity and the pyruvic acid productivity were both high, it was best to culture the strain in a medium containing 1 % Polypepton, 0.2 % glucose, 3 μg/1 dl-lipoic acid, 0.05 % l-tryptophan, and mineral salts. The optimum composition of the reaction mixture for the pyruvic acid production by the washed cells was established. Under these conditions, 17 g/1 of pyruvic acid was accumulated from 5 % glucose after 36 hr of incubation. Thus, the conversion of the pyruvic acid to tryptophan was done by adding indole, NH₄C1, pyridoxal-5′-phosphate, Triton X-100, and KOH to adjust the pH to 9.0 to the above reaction mixture. As a result, the pyruvic acid was rapidly converted to tryptophan, and the concentration of 14 g/1 was obtained after 36 hr (total 72 hr).     

The ability of selected bacterial isolates to utilise components of synthetic metal-working fluids as sole sources of carbon and nitrogen for growth

A total of 24 bacterial isolates able to grow on metal-working fluids were obtained from soil or metal-working fluids (both in-use and heavily contaminated fluids). Pure cultures of the isolates were tested for their ability to degrade a selection of components, including borate esters, phosphate ester, biocide and triethanolamine, typically found in synthetic metal-working fluids. All components, when present at a level equivalent to half that found in an in-use metal-working fluid, supported growth when utilised as the sole source of carbon and/or nitrogen. Each component was degraded by at least 50% by an individual isolate within 120 hours in batch liquid culture.     

Overproduction and crystallization of tryptophanase from recombinant cells of Escherichia coli

We have cloned the tryptophanase structural gene from Escherichia coli B/1t7-A into E. coli K-12 MD55 with a vector plasmid, pBR322. The cloned cells produced a large amount of the enzyme corresponding to more than 30% of the total soluble protein. With the enzyme obtained by this overproduction system, we have prepared three different crystals of tryptophanase, apo-enzyme, holo-enzyme, and a complex of holo-enzyme and L-alanine, by using polyethylene glycol 4000 or potassium phosphate as a precipitant and the hanging drop method. These single crystals appeared to be suitable for X-ray diffraction analysis.     

The PGE2 vaginal film: An alternative to conventional induction in multiparae with poor cervical scores

In a study involving 50 multiparous subjects with poor cervical scores (⪕3), induction of labour by conventional amniotomy and oxytocin was compared with preinduction cervical ripening using a single administration of prostaglandin E₂ (850ug) in a new vaginal film formulation. Indications for elective delivery, maternal characteristics and distribution of cervical scores in the two groups were similar. Significant changes in mean cervical score were achieved within 12 hours of film insertion. In this group, 11 subjects (45.8%) established labour within 12 hours and a further 8(33.3%) did so before 24 hours so that only 5 cases required amniotomy and oxytocin. Instrumental delivery was less in this group and none of these subjects required Caesarean section for a failure of induction. No adverse maternal or fetal side effects were observed. Convenience, ease of administration and stability of this new prostaglandin formulation make it a useful alternative to conventional induction of labour in the multiparous patient with a poor cervical score.     

Evaluation of L-Cystine-amended H2S test for the detection of faecal pollution in potable water: comparison with standard multiple tube fermentation method

L-Cystine-amended H₂S strip test was compared with conventional multiple tube fermentation tests for detecting faecal pollution in 200 drinking water samples drawn from different water sources. Of these, 134 samples were positive for total coliforms (TC), whereas the cystine-amended H₂S test was positive with 116 samples. Faecal coliforms were present in 99 samples. Seventy-seven samples were also tested with presence–absence (P–A) strip test. By taking only the colour change (acid production) as positive indication for the presence of coliforms, 12 (47%) out of 26 water samples were graded as polluted in the absence of detectable coliforms. This indicated the critical importance of observing P–A bottle for gas production along with the colour. This aspect is important when commercially available P–A bottles are used in rural areas by unskilled personnel. Among different unpiped water sources tested, 94% samples drawn from India Mark II hand-pumps had TC counts within WHO guidelines. This revised version was published online in July 2006 with corrections to the Cover Date.     

Prediction of human acute toxicity by the hep G2/24-hour/total protein assay, with protein measurement by the CBQCA method

In our previously described Hep G2/24-hour/total protein assay, protein levels were measured by using the Lowry method. This assay was the best acute in vitro assay for the prediction of human toxicity within the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) study. In order to increase the MEIC data-base with a wider range of chemicals, we were interested in introducing the more practical 3-(4-carboxybenzoyl)- quinoline-2-carboxaldehyde (CBQCA) method for the quantification of the total protein content. Therefore, we investigated whether the same good results for the prediction of acute human toxicity would be obtained with the CBQCA method. The cells were treated for 24 hours, then cytotoxicity was determined by measuring the total protein content with CBQCA. The results were quantified by using the PI50c: the concentration (in mM) of test compound required to reduce the total protein content measured with the CBQCA-method by 50% as compared to the control cells. The results were compared with the PI50, the corresponding value when the Lowry method was used. A relatively low correlation was observed between PI50 and PI50c, reflecting the large and unexpected, differences when using the two protein assays. However, when comparing the log PI50c with the human toxicity, a correlation coefficient of r(2) = 0.761 (n = 44) was obtained for exactly the same series of MEIC chemicals. This value is clearly higher than that for the Lowry method (r(2) = 0.695). Compared to the Lowry method originally used, the Hep G2/24-hour/CBQCA total protein assay has the additional important advantage that it can be very easily adapted for large-scale analyses with robotic systems, including the on-line calculation of the results.     

Laminaria augmentation of intra-amniotic prostaglandin F2α for the induction of mid-trimester abortion

From interpretation of 24-hour dose-response curves, it is improbable that mid-trimester abortion rates greater than about 80% can be accomplished with any one dose schedule of Prostaglandin F2α (PGF2α). To determine whether augmentation of intra-amniotic PGF2α with laminaria would improve the abortion rate, the results of a group of 22 gravidas treated with intra-amniotic PGF2α were compared to those of a group of 21 subjects treated with laminaria and an identical dose schedule of PGF2α. Patients with laminaria not only had a shorter mean abortion time (14.6 hours), but 95% aborted within 24 hours and all patients aborted within 24.5 hours of the initial PGF2α injection. Patients without laminaria had a longer mean abortion time (18.9 hours); only 68% aborted within 24 hours and one failed to abort within the 48-hour trial period. No significant differences in the frequency or severity of complications between the two groups were observed. Uterine contractility over the initial 6 hours of the induction was similar in the two groups. Therefore, augmenting the intra-amniotic PGF2α method with laminaria appears practicable.     

Cell-free synthesis of tryptophanase from Escherichia coli. Use of ribonucleic acid isolated from induced cells and a comparison of the product from a system employing ribosomes with that from one employing ribosomes and exogenous ribonucleic acid

1. Polyribosomes and RNA were isolated from cultures in which tryptophanase (EC 4.2.1.-) was induced. The polyribosomes were incubated under conditions of protein synthesis, in the presence of a radioactive amino acid and a post-ribosomal supernatant fraction obtained from repressed cells. The RNA preparations were incubated under conditions of protein synthesis in the presence of a radioactive amino acid and a supernatant fraction containing ribosomes from repressed cells. 2. The system was characterized and the synthesis of a radioactive protein with the same chromatographic properties as tryptophanase was demonstrated. This synthesis was shown to be time-dependent and required the presence of RNA from induced cultures, ribosomes and an energy supply; it was inhibited by chloramphenicol. 3. The maximum activity for the synthesis of this protein was found to be associated with 23S rRNA isolated from sucrose gradients. 4. The N-terminal amino acid of tryptophanase was labelled in the protein synthesized in this system but not in the protein synthesized by polyribosomes (without added RNA). Conversely, the C-terminal amino acid of tryptophanase was labelled in the polyribosome system but not in the RNA-containing system. 5. Tryptic digests of protein labelled in vitro were compared with those of tryptophanase. No labelled tryptic peptides were identified other than tryptophanase tryptic peptides. An analysis of the results implied that in the polyribosome system almost the complete tryptophanase subunit chain was labelled but that in the RNA-containing system these chains were incompletely synthesized. 6. Sucrose-gradient analysis of protein synthesized in the RNA-containing system suggested that it cannot be converted into structures with the same sedimentation properties as native tryptophanase. 7. The significance of these results for the assay of tryptophanase mRNA and for an understanding of the control of the translation of this mRNA in vivo is discussed.     

Once-daily sustained-release matrix tablets of nicorandil: Formulation and in vitro evaluation

The objective of the present study was to develop once-daily sustained-release matrix tablets of nicorandil, a novel potassium channel opener used in cardiovascular diseases. The tablets were prepared by the wet granulation method. Ethanolic solutions of ethylcellulose (EC), Eudragit RL-100, Eudragit RS-100, and polyvinylpyrrolidone were used as granulating agents along with hydrophilic matrix materials like hydroxypropyl methylcellulose (HPMC), sodium carboxymethylcellulose, and sodium alginate. The granules were evaluated for angle of repose, bulk density, compressibility index, total porosity, and drug content. The tablets were subjected to thickness, diameter, weight variation test, drug content, hardness, friability, and in vitro release studies. The granules showed satisfactory flow properties, compressibility, and drug content. All the tablet formulations showed acceptable pharmacotechnical properties and complied with in-house specifications for tested parameters. According to the theoretical release profile calculation, a oncedaily sustained-release formulation should release 5.92 mg of nicorandil in 1 hour, like conventional tablets, and 3.21 mg per hour up to 24 hours. The results of dissolution studies indicated that formulation F-I (drug-to-HPMC, 1∶4; ethanol as granulating agent) could extend the drug release up to 24 hours. In the further formulation development process, F-IX (drug-to-HPMC, 1∶4; EC 4% wt/vol as granulating agent), the most successful formulation of the study, exhibited satisfactory drug release in the initial hours, and the total release pattern was very close to the theoretical release profile. All the formulations (except F-IX) exhibited diffusion-dominated drug release. The mechanism of drug release from F-IX was diffusion coupled with erosion.     

Molecular identification of Salmonella isolates from poultry and meat productsin Irbid City,Jordan

The sensitivity and accuracy of molecular diagnosis of Salmonella from meat and poultry products using polymerase chain reaction (PCR) was compared with conventional microbiological methods. A total of 212 samples representing the most frequently used fresh and frozen meat and poultry products (whole, cut, ground, and processed) were collected from different locations within the city of Irbid. DNA was extracted directly from each food sample and amplified using Salmonella-specific primers. Samples were also analysed using conventional microbiological methods for the presence of Salmonella spp. Results showed that Salmonella was detected in 185 samples out of 212 (87%) by PCR technique, while 172 (81%) samples were detected Salmonella positive by conventional microbiological methods. On the other hand, 27 (12.7%) samples were negative by PCR and 40 (18.8%) samples were negative by conventional microbiological methods. PCR assay proved to be an effective method for Salmonella detection in meat and poultry products with high specificity and sensitivity and more importantly a less time-consuming procedure. Using PCR, Salmonella spp. detection could be achieved within 24–36 h compared to 3–8 days for the conventional microbiological methods.     

Nitrogenase activity, nodule respiration, and o(2) permeability following detopping of alfalfa and birdsfoot trefoil

Gas exchange measurements and noninvasive leghemoglobin (Lb) spectrophotometry (nodule oximetry) were used to monitor nodule responses to shoot removal in alfalfa (Medicago sativa L. cv Weevlchek) and birdsfoot trefoil (Lotus corniculatus L. cv Fergus). In each species, total nitrogenase activity, measured as H₂ evolution in Ar:O₂ (80:20), decreased to <50% of the initial rate within 1 hour after detopping, and net CO₂ production decreased to about 65% of the initial value. In a separate experiment in which nodule oximetry was used, nodule O₂ permeability decreased 50% within 5 hours in each species. A similar decrease in the O₂-saturated respiration rate (V_max) for the nodule central zone occurred within 5 hours in birdsfoot trefoil, but only after 24 hours in alfalfa. Lb concentration, also measured by oximetry, decreased after 48 to 72 hours. The decrease in permeability preceded the decrease in V_max in each species. V_max may depend mainly on carbohydrate availability in the nodule. If so, then the decrease in permeability could not have been triggered by decreasing carbohydrate availability. Both oximetry and gas exchange data were consistent with the hypothesis that, for the cultivars tested, carbohydrate availability decreased more rapidly in birdsfoot trefoil than in alfalfa nodules. Fractional Lb oxygenation (initially about 0.15) decreased during the first 24 hours after detopping but subsequently increased to >0.65 for a majority of nodules of each species. This increase could lead to O₂ inactivation of nitrogenase.     

Isolation of an Escherichia coil strain mutant unable to form biofilm on polystyrene and to adhere to human pneumocyte cells: involvement of tryptophanase

Escherichia coli adherence to biotic and abiotic surfaces constitutes the first step of infection by promoting colonization and biofilm formation. The aim of this study was to gain a better understanding of the relationship between E. coli adherence to different biotic surfaces and biofilm formation on abiotic surfaces. We isolated mutants defective in A549 pneumocyte cells adherence, fibronectin adherence, and biofilm formation by random transposition mutagenesis and sequential passages over A549 cell monolayers. Among the 97 mutants tested, 80 were decreased in biofilm formation, 8 were decreased in A549 cells adherence, 7 were decreased in their adherence to fibronectin, and 17 had no perturbations in either of the three phenotypes. We observed a correlation between adherence to fibronectin or A549 cells and biofilm formation, indicating that biotic adhesive factors are involved in biofilm formation by E. coli. Molecular analysis of the mutants revealed that a transposon insertion in the tnaA gene encoding for tryptophanase was associated with a decrease in both A549 cells adherence and biofilm formation by E. coli. The complementation of the tnaA mutant with plasmid-located wild-type tnaA restored the tryptophanase activity, epithelial cells adherence, and biofilm formation on polystyrene. The possible mechanism of tryptophanase involvement in E. coli adherence and biofilm formation is discussed.     

A simple procedure for quick identification of tryptophanase positive recombinant clones and tryptophanase regulatory mutants of bacteria

Summary A simple and rapid technique for quick identification of tryptophanase regulatory mutants and tryptophanase positive clones in a bacterial population is described. This method was used for the detection of tryptophanase regulatory mutants of Vibrio cholerae and tryptophanase positive recombinant clones of Escherichia coli.     

Early Responses of Sodium-Deficient Amaranthus tricolor L. Plants to Sodium Application

Effects of sodium application on sodium-deficient Amaranthus tricolor L. cv Tricolor seedlings were studied. Thirty-day-old A. tricolor seedlings grown without sodium received either 0.5 millimolar of NaCl or KCl, and the changes in the growth rate, chlorophyll concentration, photosynthetic oxygen evolution, and dark-oxygen consumption, and some enzyme activities were compared. Following the sodium treatment, the sodium concentration in the leaves increased from the initial value of 0.4 millimolar to 2 to 3 millimolar within 24 hours, and also the relative growth rate and O₂ evolution were enhanced within 24 hours. The stimulation of O₂ evolution was greater in the upper leaves than in the lower leaves. Although total chlorophyll concentration did not increase significantly, the increase in the chlorophyll a/b ratio was apparent within 24 hours. There were not significant increases in the C₄ photosynthetic enzyme activities; however, nitrate reductase activity increased by 350% by the sodium treatment within 24 hours, and this increase is considered not to be one of the consequences of the improved photosynthesis. Results suggest that the sodium treatment promoted CO₂ and nitrate assimilation resulting in the growth enhancement, and that sodium can be involved in some other functions than C₄ photosynthesis in A. tricolor plants.