Evaluation of a commercial beta-glucuronidase test for the rapid and economical identification of Escherichia coli

A commercial beta-glucuronidase (beta-GUR) test for the rapid and economical identification of Escherichia coli was evaluated. A total of 762 clinical strains and 228 environmental isolates were studied. More than 95% of the E. coli strains were found to be beta-GUR positive. Thirty-one clinical isolates of Shigella sonnei, 10 of Enterobacter cloacae, eight of Enterobacter aerogenes, nine of Citrobacter freundii and one of Salmonella enteritidis also gave positive results. The enzyme beta-GUR was also detected in two environmental strains of E. cloacae and one C. freundii. A comparative study between the beta-GUR test and the conventional identification system was carried out in 233 consecutive isolates of lactose positive enterobacteria. Agreement was observed in 223 cases and 190 E. coli strains were correctly identified using this test. Discrepancies were found in 10 cases: nine E. coli were beta-GUR negative and one C. freundii was beta-GUR positive. Escherichia coli was the only species positive for both beta-GUR and indole tests. This procedure permits a rapid, easy, precise and inexpensive identification of E. coli. beta-GUR positive Enterobacter strains have not previously been described.     

A rapid fluorogenic method for the detection of Escherichia coli by the production of β-glucuronidase

A medium containing the fluorogenic substrate 4-methylumbelliferyl-β-d-glucuronide was developed for the isolation and identification of Escherichia coli within 7·5 h and was based on the detection of β-glucuronidase. Optimum conditions for the rapid development of fluorescent colonies were determined. The optimum temperature was 41·5°C. Development of fluorescence was delayed when carbohydrates were incorporated into the medium. Water samples were used to evaluate the medium by surface plating and membrane filtration. The frequency of false-negative results was 6·1% and false-positives were 3·7% for freshwater samples. The false-positive organisms were identified as Klebsiella spp. and Shigella sonnei. The potential applications of the medium are discussed.     

Specific detection of Escherichia coli and Shigella species using fragments of genes coding for β-glucuronidase

Abstract The occurrence of β-glucuronidase activity, a main characteristic of Escherichia coli and the presence of the uid chromosomal region of E. coli , coding for this enzyme, were tested on representative members of enteric bacteria. DNA hybridization techniques using uid probes and ampplification experiments of uidA gene by the polymerase chain reaction (PCR) confirmed the specificity of uid genes fro E. coli and Shigella spp. (i.e., S. boydii, S. dysenteriae, S. flexneri and S. sonnei ), independent of the β-glucuronidase phenotype of bacterial strains. This specificity seemed to be conserved when studies were extended to a wide range of bacteria. It was not possible to distinguish E. coli from Shigella spp. The detection sensitivity using double stranded DNA radiolabeled probes was 3 × 10⁴ bacteria and could be brought down to 8 bacteria by PCR. Thus, the uid genes appeared to be ideal candidates for DNA probes technology to detect E. coli-Shigella species.     

Comparison of indole production and β-glucuronidase activity for the detection of Escherichia coli in a membrane filtration method

In a membrane filter method for the enumeration of Escherichia coli in water samples, the James' indole reagent has several advantages over the commonly used diaminobenzaldehyde (DAB) reagent. Results with James' reagent were easier to read because the red colour of positive colonies was more intensive and developed within a few minutes without exposure to UV light. DAB-coloured colonies were pale pink with a diffuse pink zone surrounding the colonies after 30 min of exposure to UV-source radiation. Incorporation of 4-methylumbelliferyl- β -D-glucuronide (MUG) into the selective medium to detect E. coli by means of β -glucuronidase-activity gave discouraging results. Fluorescence was difficult to read on membrane filters incubated on this medium and 14% of E. coli strains were β -glucuronidase-negative.     

A medium detecting β-glucuronidase for the simultaneous membrane filtration enumeration of Escherichia coli and coliforms from drinking water

A medium for the single membrane enumeration of Escherichia coli and coliforms from potable water was developed by the modification of the standard UK membrane filtration medium. The medium, membrane-Lactose Glucuronide Agar (m-LGA), employs a chromogenic substrate for the detection of β-glucuronidase activity and sodium pyruvate to enhance recovery of chlorine-stressed coliforms. Escherichia coli identification was significantly improved on m-LGA with 98.6% of presumptive isolates confirming. Recovery of coliforms from drinking water samples was also significantly improved.     

Observations on the use of a medium detecting β-glucuronidase activity and lactose fermentation for the simultaneous detection of Escherichia coli and coliforms

Three hundred and forty clinical isolates of Candida species and Saccharomyces cerevisiae were tested in order to evaluate different methods for identification of Candida albicans using fluorogenic or chromogenic substrates. Detection of N -acetyl-β-D-galactosaminidase (NAGase) was performed with ready-to-use agars such as Fluoroplate Candida Agar (Merck, Germany), MUAG Candida Agar and MUAG Sabouraud Agar (Biolife, Italy) which contained 4-methylumbelliferyl- N -acetyl-β-D-galactosaminide (4-MUAG). MUAG Candi Kit and RAP-ID-ALBICANS (Biolife, Italy) and Albicans ID Agar (bioMérieux, France) were also used. The Vitek AMS System was used as a reference identification method for all isolates. NAGase activity could be detected for C albicans with Fluoroplate Candida Agar (98.8%), MUAG Sabouraud Agar (98.4%), Albicans ID (99.6%). MUAG Candi Kit (97.5%) and RAP-ID-ALBICANS (96.2%). Proline aminopeptidase examined with RAP-ID-ALBICANS was present in 98.7% of C. albicans. There was one false-positive result for C. tropicalis (9.1%) on Fluoroplate Candida Agar, one false-positive result for C. glabrata (2.2%) on Albicans ID Agar: five false-negative results for C. albicans (3.1%), but no false-positive results for the other tested species were observed with RAP-ID-ALBICANS.     

Influence of various bile salts on ββ-glucuronidase activity of intestinal bacteria

While the decrease of the β-glucuronidase activity of sonicated cells of Clostridium perfringens and Escherichia coli was obvious for sodium deoxycholate (DC), it was not so obvious for other bile salts (sodium glycocholate and sodium cholate). The enzyme activity of intact cells of these bacteria was significantly enhanced by the presence of DC, but not by the other bile salts in the buffer. These results suggest that the permeability of the bacterial cells is increased more by the presence of DC than by other bile salts.     

Effect of seawater on Escherichia coli β-galactosidase activity

An investigation of β-galactosidase activity of Escherichia coli strain H10407, under different physiological and environmental conditions, e.g. induced and uninduced osmotic stress, light, etc., was undertaken. In this study E. coli was employed as a model for faecal coliforms in waste water. β-Galactosidase activity was induced by isopropyl-β-D-thiogalactoside (IPTG). Enzyme activity (U cell^-1)/cell for sewage bacteria and for induced E. coli was similar, i.e. log U cell^-1= -8.5 whereas uninduced E. coli yielded log U cell^-1= -12.1. Initial enzyme activity was not dependent on phase of growth of the cell (exponential vs stationary phase) or whether marine or fresh water at the time of initial dilution. However, osmotic change resulted in a decrease in culturable cells, even though enzyme activity remained constant. A significant decrease in the number of culturable bacteria, followed by a decrease in β-galactosidase activity, was observed after exposure of cells to visible light radiation. It is concluded that β-galactosidase enzyme is retained in viable but non-culturable E. coli. Furthermore, β-galactosidase appears to offer a useful and rapid (25 min) measure of the viability of faecal coliforms, and therefore, of the water quality of bathing and shellfishing areas.     

A note on starch hydrolysis and β-glucuronidase activity among flavobacteria

Most flavobacteria tested with the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide possessed β-glucuronidase (GUD), but when some of the same strains were tested with the API ZYM gallery, all were negative for GUD. Conflicting reports also appear in the literature about starch hydrolysis among flavobacteria. We observed that the results obtained can depend on the medium used and the length of incubation. Our results indicate that GUD activity and starch hydrolysis are more widely distributed in the genus Flavobacterium than previously reported.     

Influence of bile salts on β-glucuronidase activity of intestinal bacteria

T. FUJISAWA AND M. MORI. 1996. The β-glucuronidase activity of intact cells of Escherichia coli and Clostridium perfringens was increased in the presence of bile salts. In contrast, bile salts had inhibitory effects on the activity of β-glucuronidase extracted from the lysed cells. These results suggest that the permeability of the bacterial cells is increased by the presence of bile salts, and that bile salts may significantly enhance bacterial β-glucuronidase activity in the intestinal tract.     

A note on lactobacilli and β-glucuronidase activity in the intestinal contents of mice

A comparison was made of caecal β-glucuronidase activity in the caecal contents of mice with or without lactobacilli as members of the normal microflora. Male mice that did not have lactobacilli as intestinal inhabitants had approximately 52% more caecal β-glucuronidase activity than their female counterparts. Colonization of male mice by lactobacilli reduced the β-glucuronidase activity to that of female mice.     

Effect of chlorination on β-D-galactosidase activity of sewage bacteria and Escherichia coli

The effect of chlorine on β- D- galactosidase activity of sewage bacteria and Escherichia coli was studied. β- D- galactosidase activity of sewage was more resistant to chlorine than faecal coliform cultivability. At low initial dosage (0·05 mg Cl₂ l⁻¹) neither cultivability (colony-forming units (cfu)), nor enzyme activity of E. coli suspensions were severely impaired. When initial chlorine concentration was increased to 0·1 mg Cl₂ l⁻¹, the cfu number decreased whereas enzyme activity remained high, i.e. the enzyme activity calculated cfu⁻¹ increased. At higher chlorine doses both cfu and enzyme activity were reduced, but non-cultivable cells retained assayable activity after chlorination. Mean values of the enzyme activity calculated cfu⁻¹ decreased when the chlorine dosage was increased from 0·1 to 0·5 mg Cl₂ l⁻¹, but were not significantly different ( P > 0·05) for dosages of 0·2–0·7 mg Cl₂ l⁻¹. After chlorination, β- D- galactosidase activity of E. coli was less reduced than cfu and direct viable count numbers, but more reduced than 5-cyano-2-3, ditolyl tetrazolium chloride and total cell counts, and the enzyme activity represented an alternative activity parameter of chlorinated samples.     

Seric and secretory antibodies reactive to α, β and γ intimins of Escherichia coli in healthy Brazilian adults

Intimin is essential for attaching and effacing lesions by pathogens such as enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC), and the antigenic polymorphism of intimin determines distinct subtypes. Our aim was to investigate the presence of immunoglobulin G (IgG) and IgA antibodies reactive to α, β and γ intimins in serum and colostrum from healthy Brazilian adults. We found seric IgG and secretory IgA antibodies reactive to conserved and variable regions of α, β and γ intimins and a positive correlation between the concentrations of these antibodies in both serum and colostrum that suggested cross reactivity among anti-intimin antibodies, as was confirmed by immunoblotting and absorption. The concentrations of anti-conserved region antibodies were higher than those of variable region antibodies. The presence of antibodies reactive to EHEC antigens could result from contact with EPEC or with other bacteria of the environment even though this bacterium is not frequent in Brazil, and suggests possible protection against EHEC.     

Isolation and preliminary characterization of β-lactamase excretory mutants of Escherichia coli K-12

Abstract Escherichia coli exc mutants able to release the plasmid pBR322-encoded β-lactamase (EC 3.5.2.6) into the extracellular medium have been isolated using a new in situ plate assay.
A preliminary characterization of the exc mutants was carried out: the presence of exc mutations was associated with a specific or pleiotropic pattern of excretion of periplasmic enzymes, an increased sensitivity to different growth inhibitors (EDTA, chloramphenicol, cholic acid) and a poor growth on various carbon sources.
After quantitative analysis, three groups of exc mutants were identified on the basis of their temperature-dependent or -independent pattern of growth and β-lactamase synthesis and excretion.     

Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the ααααααα/βββββββ-type protect spores against this DNA damage

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed α⁻β⁻) lacking the two major α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and α⁻β⁻ spores to formaldehyde treatment, which caused significant expression of a recA - lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross-linking in both wild-type and α⁻β⁻ spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.     

Low copy number vectors for expression of fused genes to β-galactosidase in Escherichia coli

A series of six expression vectors, pXM184Lac.A, B, C, pXM184Z.A, B, C, based on the low copy plasmid pACYC184 that allow for expression of proteins fused to beta-galactosidase in Escherichia coli is described. A level of 50,000 units of beta-galactosidase is routinely observed and is easily identifiable on protein gels. This paper also reports the tight regulation of expression of the Trc promoter in these vectors using the LacIq repressor.