Detection of false-positives among total and fecal coliform counts by factorial analysis of correspondence

Application of an analysis of correspondence to the biochemical characteristics of total and fecal coliforms isolated in the Ivory Coast permitted us to separate two small clusters of isolates different from the main clusters, which included isolates from human and animal feces. The isolates grouped in the small clusters were from water samples. An analysis of the biochemical characteristics which permitted the segregation of the "water-specific" isolates from the main clusters indicates that water-specific total coliforms were citrate positive, indole negative, and amygdaline positive. Water-specific fecal coliforms were either citrate positive, indole negative, amygdaline positive, and inositol negative or indole negative, amygdaline positive, and inositol positive. Any isolates not fitting the above patterns could be considered of fecal origin. If this observation is confirmed under temperate climates and for a greater number of isolates, these simple tests could be used to confirm the fecal origin of coliforms.     

Detection of false-positives among total and fecal coliform counts by factorial analysis of correspondence.

Application of an analysis of correspondence to the biochemical characteristics of total and fecal coliforms isolated in the Ivory Coast permitted us to separate two small clusters of isolates different from the main clusters, which included isolates from human and animal feces. The isolates grouped in the small clusters were from water samples. An analysis of the biochemical characteristics which permitted the segregation of the "water-specific" isolates from the main clusters indicates that water-specific total coliforms were citrate positive, indole negative, and amygdaline positive. Water-specific fecal coliforms were either citrate positive, indole negative, amygdaline positive, and inositol negative or indole negative, amygdaline positive, and inositol positive. Any isolates not fitting the above patterns could be considered of fecal origin. If this observation is confirmed under temperate climates and for a greater number of isolates, these simple tests could be used to confirm the fecal origin of coliforms.     

沙门氏菌Ⅱ的三个新血清型

Eight cultures isolated from intestinal contents of reptiles were belonged to 3 new serotypes of Salmonella. They were all ducitol fermented, malonate utilized, but not attack lactose and salicin, no growth in KCN broth, ONPG negative. Therefore, they would be included in Salmonella II. They were all attacked by Felix phage O-I. Three represented strains were selected for antigen analysis. Their antigenic formula were identified as follows: S3194 Salmonella II 6,7:1,v:e,n,z15 S3196 Salmonella II 6, 7:y: e, n, z(1)5 S3195 Salmonella II 6, 8: e, h: 1,2 Among them, S3196 was indole positive belonging to a rare biotype. In addition, there were two other cultures as well as the formula of S3194, and three other cultures as well as the formula of S3196 (one of indole positive, two of indole negative).     

THE RAPID IDENTIFICATION OF ESCHERICHIA COLI I BY THE PRODUCTION AT 44° OF BOTH INDOLE AND GAS FROM LACTOSE

SUMMARY: The modification of the 44° test proposed by Mackenzie, Taylor & Gilbert (1948) is useful for the rapid identification of Escherichia coli I in water and foods. False positive tests caused by other coli-aerogenes bacteria, or by their association with other organisms, can be considered rare. Only a few E. coli I fail to produce indole or ferment lactose at 44°, and further confirmation for routine purposes is necessary only when the results at 44° are discordant, e.g. indole positive-lactose negative or indole negative-lactose positive.     

STUDIES OF THE DETERMINATION OF THE BACTERIUM COLI TYPE I CONTENT OF FARM WATER SUPPLIES

SUMMARY: A – comparison of the suitability of brilliant green bile broth and MacConkey's broth at 44° for the detection of Bacterium coli type I in farm water supplies, showed that 83.1% of the samples had no difference in the number of positive tubes at 44°, and only 5 samples (1.7%) had a significantly higher number of positive tubes in MacConkey's broth.
Of 707 strains of coli-aerogenes bacteria isolated from 44° positive tubes of both media, 94.5% were Bact. coli type I. Strains of Bact. coli type II and Bact. aerogenes type I which were 44° positive constituted 3.7% and 0.4% respectively, all of which were indole negative at 44°. In addition there were 10 strains (1.4%) of 44° positive Intermediate type II, 9 of which were indole positive at 44°.
An appreciable number (6.6%) of Bact. coli type I strains failed to give a positive indole reaction in 24 hr at 44°.     

Escherichia coli variants for gas and indole production at elevated incubation temperatures.

Two strains of Escherichia coli were subjected to heat and cold-storage treatments to determine the stability of the fecal E. coli characteristics of gas production from lactose and indole production at elevated incubation temperatures. No variants were detected with repeated sublethal heat treatment. A high incidence of variants was observed with extended cold storage of the organisms in liquid and semisolid media, especially with poor nutrient composition, and in the absence of cryoprotective agents. The indole characteristic at elevated temperature was more stable than the production of gas from lactose. The critical temperature at which both gas production from lactose and the indole characteristic were lost was 44.5 degrees C. It appeared that the variants resulted from increased temperature sensitivity of the formic hydrogen lyase and tryptophanase enzymes, respectively.     

Escherichia coli variants for gas and indole production at elevated incubation temperatures

Two strains of Escherichia coli were subjected to heat and cold-storage treatments to determine the stability of the fecal E. coli characteristics of gas production from lactose and indole production at elevated incubation temperatures. No variants were detected with repeated sublethal heat treatment. A high incidence of variants was observed with extended cold storage of the organisms in liquid and semisolid media, especially with poor nutrient composition, and in the absence of cryoprotective agents. The indole characteristic at elevated temperature was more stable than the production of gas from lactose. The critical temperature at which both gas production from lactose and the indole characteristic were lost was 44.5 degrees C. It appeared that the variants resulted from increased temperature sensitivity of the formic hydrogen lyase and tryptophanase enzymes, respectively.     

Energetic adaptation of ligand binding to subunit structure of tryptophan synthase from Escherichia coli

The binding of indole and L-serine to the isolated alpha and beta 2 subunits and the native alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli was investigated by direct microcalorimetry to reveal the energetic adaptation of ligand binding to the subunit structure of a multienzyme complex. In contrast to the general finding that negative heat capacity changes are associated with ligand binding to proteins, complex formation of indole and the alpha subunit involves a small positive change in heat capacity. This unusual result was considered as being indicative of a loosening of the protein structure. Such an interpretation is in good agreement with results of chemical accessibility studies (Freedberg & Hardman, 1971). Whereas the thermodynamic parameters of indole binding are not influenced by the subunit interaction, the large negative change in heat capacity of -6.5 kJ/(K X mol of beta 2) measured for the binding of L-serine to the isolated beta 2 subunit disappears completely when serine interacts with the tetrameric complex. These data demonstrate that the energy transduction pattern and therefore the functional roles of the substrates indole and L-serine vary strongly with the subunit structure of tryptophan synthase.     

Thy-1 variants of mouse lymphomas: biochemical characterization of the genetic defect

The surface proteins of Thy-1 positive mouse lymphomas and Thy-1 negative variants were labeled by lactoperoxidase-catalyzed iodination and characterized by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate and by immunological studies. The loss of the serologically defined Thy-1 antigenic determinant correlated with the absence of a radioactive band corresponding to a T lymphocyte-specific surface glycoprotein, T25, on autoradiographs of the iodinated proteins of Thy-1 negative variants. Reexpression of Thy-1 on hybrid cells derived from fusions of complementary Thy-1 negative variants correlated with the reappearance of T25. Quantitative absorption studies using an antiserum which specifically recognized T25 confirmed that the Thy-1 negative variants have no detectable T25 (<0.03 of that of the Thy-1 positive lines) on their surface.Biosynthetic labeling studies revealed that none of the Thy-1 negative variants synthesized T25. However, synthesis of an immunologically cross-reactive molecule could be detected in two variants. On the basis of these results, we propose a model which describes a possible structure of the Thy-1 antigenic determinant and explains the biochemical nature of the genetic lesions leading to the loss of Thy-1 in these variants.     

Ultrastructure of Nocardia-like variants of Mycobacterium smegmatis and chemical composition of the basal cell wall layer

Mycobacterium smegmatis, its orange-red--pigmented (OR) variants, and back mutant strains were examined by electron microscopy using ultrathin sectioning, negative or positive staining, and freeze-fracture-etching methods. The parental and back mutant strains showed almost identical ultrastructures. Specifically, thick ramified fibers measuring about 15 nm in diameter were always visible in the positively stained cell wall, although they were not readily visualized with negative staining or freeze-fracture-etching. In contrast, the cell walls of OR variants contained fibrous networks measuring about 11 nm in diameter, which could be observed by positive and negative staining as well as freeze-fracture-etching. Although cytoplasmic structures appeared similar among the four strains examined, mesosomes were significantly more abundant in the OR variants. The basal layer of the cell wall obtained as a phenol residue consisted of a dense membranous matrix containing scattered fibrous structures in the parental and back mutant strains, and fibrous networks in the OR variants. Chemical analyses showed that the basal layers of all four strains contained the same neutral sugars, amino sugars, and amino acids, i.e., arabinose, galactose, muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid. The alpha-branched, beta-hydroxylated fatty acids contained in the basal layers differ among the four strains, however, with nocardomycolic acids being present in the OR variants and mycolic acids in the parental and back mutant strains. Our previous conclusion that OR variants of M. smegmatis have characteristics similar to those of nocardia is supported by the present study.     

Confirmation of Escherichia coli and its distinction from Klebsiella species by gas and indole formation at 44 and 44.5 degrees C

AIMS: In the enumeration of coliform bacteria, confirmation of Escherichia coli has been based upon gas and indole production at the elevated incubation temperature. The test for gas production has recently been questioned. The aim of this study was to investigate the impact of gas production test on the reliability of confirmation of E. coli. METHODS AND RESULTS: The impact of several media on growth, gas and/or indole formation was tested at 44 and 44.5 degrees C using 547 environmental isolates. These were mainly E. coli, Klebsiella pneumoniae, K. oxytoca and Enterobacter cloacae strains. Another set of 250 faecal and environmental klebsiellae were tested for their maximum temperature for growth (Tmax) and for gas formation. Escherichia coli and even K. pneumoniae grew well in all the media, but gas production was more dependent on the medium used. Growth of the mainly gas negative Ent. cloacae and K. oxytoca strains was still more sensitive to the medium and incubation conditions. Tryptophan salt broth was the most productive medium for the indole test, followed by lauryl tryptose mannitole and tryptone mannitol ricinoleate broth (TRM). Tmax of K. oxytoca was clearly lower than Tmax of K. pneumoniae but a rather high fraction of its isolates produced indole at 44.5 degrees C. CONCLUSIONS: False-positive E. coli confirmation is possible if gas production is not tested for and the confirmation is based on indole test only. SIGNIFICANCE AND IMPACT OF THE STUDY: Erroneous positive results on routine analysis for E. coli can occur.     

YAC-1 MHC class I variants reveal an association between decreased NK sensitivity and increased H-2 expression after interferon treatment or in vivo passage

Two H-2 negative variants of the YAC-1 lymphoma were selected by mutagenization and sequential in vitro selections and compared with wild-type cells for changes in NK sensitivity and H-2 expression after interferon treatment or in vivo passage. The H-2 negative variants and the low H-2 expressor YAC-1 wild-type cells had similar NK sensitivity. However, IFN-beta or recombinant IFN-gamma pretreatments increased the H-2 expression of YAC-1 and protected them from NK lysis, whereas the H-2 variants, which remained H-2 negative, were not protected and often more sensitive to NK lysis. The H-2 variants were similarly susceptible as wild-type cells to three other cellular effects of interferon: protection from virus infection, modulation of Con A capping, and inhibition of cell proliferation. Thus, the only interferon-mediated effect that distinguished the H-2 negative variants from wild-type cells was the inability of the former to increase their H-2 expression and decrease their NK sensitivity. The wild-type YAC-1 line showed increased H-2 expression and decreased NK sensitivity after in vivo passage. In contrast, in vivo passaged H-2 variants showed no reexpression of H-2, and remained NK sensitive. The altered responses to interferon and in vivo passage were specific for loss or down-regulation of H-2, because Thy-1 loss (H-2 positive) YAC-1 variants behaved as the wild-type cells in all respects. This study supports the hypothesis that NK cells may function in vivo to eliminate host cells that fail to express H-2 after interferon stimulation during an immune response; such cells are a potential threat because they may escape recognition by T lymphocytes despite the expression of viral or tumor-associated antigens.     

In Vitro Susceptibility of Selected Bacteria to Cefaclor

Cefaclor is an orally absorbed cephalosporin antibiotic chemically and pharmacologically similar to cephalexin. It appears to be more active than cephalexin against susceptible strains. The in vitro sensitivity of 230 clinical bacterial isolates to cefaclor was studied. Most isolates of S. aureus, K. pneumoniae, E. coli, and indole negative Proteus species were inhibited at clinically attainable serum and urine concentrations. Like cephalexin, cefaclor was less active against isolates of Enterobacter species, indole positive Proteus species and enterococci although many of these isolates were inhibited at concentrations achievable in urine.     

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson’s disease and beta-amyloid and tau in Alzheimer’s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.     

Tryptophane-205 of human topoisomerase I is essential for camptothecin inhibition of negative but not positive supercoil removal

Positive supercoils are introduced in cellular DNA in front of and negative supercoils behind tracking polymerases. Since DNA purified from cells is normally under-wound, most studies addressing the relaxation activity of topoisomerase I have utilized negatively supercoiled plasmids. The present report compares the relaxation activity of human topoisomerase I variants on plasmids containing equal numbers of superhelical twists with opposite handedness. We demonstrate that the wild-type enzyme and mutants lacking amino acids 1–206 or 191–206, or having tryptophane-205 replaced with a glycine relax positive supercoils faster than negative supercoils under both processive and distributive conditions. In contrast to wild-type topoisomerase I, which exhibited camptothecin sensitivity during relaxation of both negative and positive supercoils, the investigated N-terminally mutated variants were sensitive to camptothecin only during removal of positive supercoils. These data suggest different mechanisms of action during removal of supercoils of opposite handedness and are consistent with a recently published simulation study [Sari and Andricioaei (2005) Nucleic Acids Res., 33, 6621–6634] suggesting flexibility in distinct parts of the enzyme during clockwise or counterclockwise strand rotation.     

Haplotype-based inference of the distribution of fitness effects

Recent genome sequencing studies with large sample sizes in humans have discovered a vast quantity of low-frequency variants, providing an important source of information to analyze how selection is acting on human genetic variation. In order to estimate the strength of natural selection acting on low-frequency variants, we have developed a likelihood-based method that uses the lengths of pairwise identity-by-state between haplotypes carrying low-frequency variants. We show that in some nonequilibrium populations (such as those that have had recent population expansions) it is possible to distinguish between positive or negative selection acting on a set of variants. With our new framework, one can infer a fixed selection intensity acting on a set of variants at a particular frequency, or a distribution of selection coefficients for standing variants and new mutations. We show an application of our method to the UK10K phased haplotype dataset of individuals.     

A modular library of small molecule signals regulates social behaviors in Caenorhabditis elegans

The nematode C. elegans is an important model for the study of social behaviors. Recent investigations have shown that a family of small molecule signals, the ascarosides, controls population density sensing and mating behavior. However, despite extensive studies of C. elegans aggregation behaviors, no intraspecific signals promoting attraction or aggregation of wild-type hermaphrodites have been identified. Using comparative metabolomics, we show that the known ascarosides are accompanied by a series of derivatives featuring a tryptophan-derived indole moiety. Behavioral assays demonstrate that these indole ascarosides serve as potent intraspecific attraction and aggregation signals for hermaphrodites, in contrast to ascarosides lacking the indole group, which are repulsive. Hermaphrodite attraction to indole ascarosides depends on the ASK amphid sensory neurons. Downstream of the ASK sensory neuron, the interneuron AIA is required for mediating attraction to indole ascarosides instead of the RMG interneurons, which previous studies have shown to integrate attraction and aggregation signals from ASK and other sensory neurons. The role of the RMG interneuron in mediating aggregation and attraction is thought to depend on the neuropeptide Y-like receptor NPR-1, because solitary and social C. elegans strains are distinguished by different npr-1 variants. We show that indole ascarosides promote attraction and aggregation in both solitary and social C. elegans strains. The identification of indole ascarosides as aggregation signals reveals unexpected complexity of social signaling in C. elegans, which appears to be based on a modular library of ascarosides integrating building blocks derived from lipid β-oxidation and amino-acid metabolism. Variation of modules results in strongly altered signaling content, as addition of a tryptophan-derived indole unit to repellent ascarosides produces strongly attractive indole ascarosides. Our findings show that the library of ascarosides represents a highly developed chemical language integrating different neurophysiological pathways to mediate social communication in C. elegans.     

Characterisation of glucosinolates using electrospray ion trap and electrospray quadrupole time-of-flight mass spectrometry

Twelve naturally occurring glucosinolates displaying alkenyl, hydroxylated, methylsulphinyl, aromatic and indole side chains were investigated by both negative and positive ion electrospray ionisation-tandem mass spectrometry (ESI-MS/MS). In order to resolve the MS/MS spectra obtained from the anion and cation molecular ions of glucosinolates, the different fragments were investigated by MSn experiments using an ion trap spectrometer. The MS3 spectra obtained permitted possible fragmentation schemes to be proposed. These were supported by accurate mass measurements of some characteristic diagnostic ions with the help of a quadrupole time-of-flight instrument. The negative ion ESI-MS/MS behaviour of the different glucosinolates investigated in this study confirmed previously described patterns and revealed new interesting structural informative fragments. Some are common to all the glucosinolates and others are highly specific for a type of variable side chain. The positive ion ESI-MS/MS fragments obtained from the [MNa+Na]+ or [MK+K]+ molecular ions did not provide complementary specific diagnostic ions. Nevertheless, when compared with the negative ion mode, the daughter ions appeared more homogenous and with a better relative abundance for all of the 12 compounds studied. Moreover, the positive ion mode appeared to be more efficient than the negative mode for the study of methoxylated glucosinolates and should be useful to detect the glucosinolates present as organic salts in crude plant extracts.     

Changes of net charge and alpha-helical content affect the pharmacokinetic properties of human serum albumin

The pharmacokinetics of 17 genetic variants of human serum albumin with single-residue mutations and their corresponding normal albumin were studied in mice. In all cases, the plasma half-life was affected, but only variants with +2 changes in charge prolonged it, whereas changes in hydrophobicity decreased it. Good positive and negative correlations were found between changes in alpha-helical content taking place in domains I+III and domain II, respectively, and changes in half-lives. No correlation was found to type of mutation or to changes in heat stability as represented by DeltaH(v). Liver and kidney uptake clearances were also modified: alpha-helical changes of domains I+III showed good negative correlations to both types of clearances, whereas changes in domain II only had a good positive correlation to kidney uptake clearance. No correlation between the other molecular changes and organ uptakes was observed. The relatively few correlations between changes in molecular characteristics and the organ uptakes of the variants are most probably due to different handling by plasma enzyme(s) and the various types of cell endocytosis. Of the latter, most lead to destruction of albumin, but at least one results in recycling of the protein. The present information should be useful when designing recombinant, therapeutical albumins or albumin products with a modified plasma half-life.