Genotypic and phenotypic analysis of Enterobacter sakazakii strains from an outbreak resulting in fatalities in a neonatal intensive care unit in France.

In 1994, an outbreak of Enterobacter sakazakii infections occurred in a neonatal intensive care unit in France from 5 May to 11 July. During the outbreak, 13 neonates were infected with E. sakazakii, resulting in 3 deaths. In addition, four symptomless neonates were colonized by E. sakazakii. The strains were subjected to 16S rRNA gene sequence analysis, genotyped using pulsed-field gel electrophoresis, and phenotyped for a range of enzyme activities. E. sakazakii was isolated from various anatomical sites, reconstituted formula, and an unopened can of powdered infant formula. A fourth neonate died from septic shock, attributed to E. sakazakii infection, during this period. However, 16S rRNA gene sequence analysis revealed that the organism was Enterobacter cloacae. There were three pulsotypes of E. sakazakii associated with infected neonates, and three neonates were infected by more than one genotype. One genotype matched isolates from unused prepared formula and unfinished formula. However, no pulsotypes matched the E. sakazakii strain recovered from an unopened can of powdered infant formula. One pulsotype was associated with the three fatal cases, and two of these isolates had extended-spectrum beta-lactamase activity. It is possible that E. sakazakii strains differ in their pathogenicities, as shown by the range of symptoms associated with each pulsotype.

Comparison of Candida ID medium with sabouraud-chloramphenicol agar for the isolation of yeasts from clinical haematology surveillance specimens.

Candida ID is a new chromogenic medium for the identification of yeasts from clinical specimens. C. albicans produces blue pigmentation, whereas pink pigmentation is produced by C. tropicalis, C lusitaniae, C. guilliermondii and C. kefyr; other Candida species appear white. In this study, 240 clinical samples (throat swabs and stool samples) from haematology patients were inoculated on to Candida ID and Sabouraud-chloramphenicol agar in parallel, yielding a total of 105 yeasts; the media had overall detection rates of 85.7% and 86.7% respectively. The sensitivity of Candida ID for identification of C. albicans by blue pigmentation was 52.9% at 24 h and 94.1% at 48 h. Specificity of the blue pigmentation was 100% at 48 h. Two strains of C. tropicalis were identified, one produced pink pigmentation at 72 h, the other strain did not produce any pigmentation after 5 days. Candida ID was superior in detecting mixtures of yeasts compared with Sabouraud-chloramphenicol agar. Candida ID is a suitable primary isolation medium for yeasts from clinical specimens, providing rapid direct identification of C. albicans and enhanced detection of mixtures.

Attitudes and use of alternative therapies in UK prostate cancer patients-isn't it time we were in the know?

With increasing media interest in prostate cancer and the availability of data to patients from support groups and the Internet, the knowledge and use of alternative therapies by patients is becoming more common. The purpose of our study was to quantify patient awareness and use of alternative therapies for the prevention and treatment of prostate cancer in the UK. In May 2000, we performed a survey of men attending our urology outpatient clinic for prostate cancer evaluation or follow-up. All men diagnosed with and those at high risk (abnormal prostate specific antigen) for prostate cancer were eligible for the study. Each eligible patient was then sent an anonymous 25-item questionnaire to explore their knowledge and use of various alternative therapies for prostate cancer. Out of 195 patients who were sent the questionnaire, 168 responded, for a response rate of 86%. One hundred and sixty-four were analysed. Eight-two out of 164 (50%) were aware of alternative therapies for prevention/treatment of prostate cancer, the most common were tomatoes/tomato-based products and low-fat diet. There were 27 (16.5%) respondents taking alternative therapies for their prostate. Private patients were more aware (60.4% private vs 46.2% NHS) of complimentary therapies and were more likely to take them (27.9% private vs 12.4% NHS) than National Health Service patients. The majority of patients (60%) had not informed their GP or urologist. Fifteen therapies and 12 medication sources were recorded. Asked if doctors should discuss non-prescribed therapies, even if there is no proven benefit, 62% said 'yes' while 29% said 'no'. Alternative therapy use for prostate cancer is likely to increase. If we don't ask patients specifically whether they are taking them, patients are unlikely to tell us. Urologists and clinical oncologists treating men with prostate cancer need to be aware of alternative therapies and have some understanding of any benefit or harm, not only to be able to answer patient's questions and offer advice, but also to consider interactions with other treatments.Prostate Cancer and Prostatic Diseases (2001) 4, 235-241.

Chromogenic urinary tract infection medium: evaluation and introduction for routine urine culture in a large clinical microbiology laboratory.

Evaluation and introduction of a chromogenic urinary tract infection (UTI) medium for the primary culture of routine urine specimens in a very large clinical microbiology laboratory is described. A modified scheme for the direct identification of the main groups of organisms causing UTI has led to quality improvements, with enhanced discrimination of mixed cultures and in efficiencies in staffing and working time that counterbalance increases in media costs. These improvements, together with the concurrent introduction of boric acid preservative for specimens from general practice, significantly reduced the level of positive results by 4.6% (2383 specimens) over an eight-month period (P <0.001). Chromogenic medium provided a reliable alternative to CLED medium for the routine culture of urine specimens.

A 23-year review of the management of acute retention of urine: progressing or regressing?

A retrospective review of all patients in Oxford under the care of one consultant urologist (GJF) who presented on alternate years over a 23-year period with acute retention of urine was undertaken. Data were collected on the: (i) number of patients discharged from hospital with an in-dwelling catheter; (ii) duration of catheter drainage prior to surgery; and (iii) duration of postoperative stay. In all, 244 patients underwent prostatectomy. Over the 23-year period, there was a significant increase in the proportion of patients discharged prior to surgery (P < 0.001) as well as their median duration of catheterisation (P < 0.001): more than 50% were catheterised for more than 3 months in 1997. Conversely, post-operative hospital stay has decreased. Prolonged catheter drainage carries considerable morbidity, with 72% experiencing some complication. Most patients feel they lose dignity, 69% consider it uncomfortable and more than 50% complain of burning sensations, bladder spasms and a persistent desire to micturate. We recommend that patients should not be placed on routine waiting lists where they are liable to remain for an unacceptably long time. Targets should be set to admit them within a set period and theatre lists made available. We feel that six weeks is a realistic target.

A comparison of two motion analysis systems for the measurement of two-dimensional rearfoot motion during walking.

The purpose of this study was to compare two-dimensional rearfoot motion during walking measured by a traditional video-based motion analysis system to that of an electromagnetic analysis system. Twenty-five individuals (15 men, 10 women) with a mean age of 29.8 years served as subjects for this study. The results of the study showed that there was a high correlation (r = 0.945) between the mean motion paths produced by the two systems, indicating that they were very similar. The electromagnetic motion analysis system was able to produce these similar results in a fraction of the time required by the video-based system.

Biological method for protection of 6-position of sucrose and its use in synthesis of disaccharide high-intensity sweetener.

A general method for protecting the 6 primary hydroxyl position of sucrose is described. It involves the production of glucose-6-acetate by fermentation of glucose using a strain of Bacillus megaterium followed by conversion to sucrose-6-acetate as a kinetic product using a specially selected fructosyl transferase producted by a newly isolated strain of Bacillus subtilis. The sucrose-6-acetate was found to be more lipophilic than expected, and this property aided its purification by chromatography. Pure sucrose-6-acetate may then be chlorinated and subsequently deacetylated to give the high-intensity sweetener 4,1',6'-trichlo-4,1',6'-trideoxygalactosucrose (sucralose) in high yields. This process involves fewer steps than are required for chemical synthesis using trityl chloride and acetic anhydride. Related intensely sweet molecules which were synthesized by similar methods included 4,1',6'-trichloro, 4,1',6'-trideoxy L-arabinosucrose, and 4,1',6'-trichloro-4,6,1',6'-tetradeoxy-galactosucrose. They were obtained from xylose and 6-deoxyglucose, respectively, via the intermediates xylsucrose and 6-deoxysucrose, formed by the reaction of the fructosyl transferase on the monosaccharide acceptors.

Biocatalytic synthesis of disaccharide high-intensity sweeterner sucralose via a tetrachlororaffinose intermediate.

A second high-yielding bioorganic synthesis of the highintensity sweetener sucralose (4,1',6'-trichloro-4,1',6'-trideoxygalactosucrose) is described. This procedure involves the chemical chlorination of raffinose to form a novel tetrachloroaffinose intermediate (6,4',1'',6''-tetrachloro-6,4',1'',6''-tetradeoxygalactoraffinose; TCR) followed by the enzymic hydrolysis of the alpha-1-6 glycosidic bond of TCR to give sucralose and 6-chlorogalactose. Commercial enzyme preparations and microorganisms were screened to select alpha-galactosidases which have high catalytic activity on this compound. The most active enzyme was produced by a strain of Mortierella vinacea and had a maximum rate of 118 mumol sucralose/g dry weight cells/h, which was approximately 5% of the activity toward raffinose, and a K(m) of 5.8 mM toward TCR. The enzyme could be used in the form of mycelial pellets in a continuous packed bed column reactor. The reaction was also studied in a water-immiscible hydrophilic organic solvent, such as methyl isobutyl ketone, to overcome the poor aqueous solubility of TCR and to increase volumetric productivity. Synthesis of raffinose was achieved from saturated aqueous solutions of galactose and sucrose using a selected alpha-galactosidase from Aspergillus niger. When raffinose is used as a starting material for sucralose synthesis, this route has fewer steps than either the preceeding method using glucose-6-acetate as an intermediate or the complete chemical synthesis from sucrose. The relative merits of the two bioorganic routes and the utility of such methods to synthesize new sugars are discussed.

Urinary tract infection with anaerobic bacteria following endoscopic urethral instrumentation.

A prospective study of the pathogenic role of anaerobic bacteria was undertaken by suprapubic bladder aspiration in 100 patients prior to routine endoscopic urethral instrumentation. Anaerobic and/or micro-aerophilic bacteria were isolated from the suprapubic specimens of 11 patients (greater than 10(5) organisms/ml from 8), with identical species growing heavily from corresponding cystoscopic specimens and/or urethral swabs. Previous cystoscopies in all 11 patients and the presence of bladder tumour at the time of sampling in six were significantly and independently associated with the isolation of anaerobic bacteria from suprapubic specimens. It was concluded that transurethral instrumentation entails a definite risk of anaerobic urinary infection in addition to the recognised risk of aerobic urinary infection. The findings suggest that the antibiotics used as prophylaxis for or treatment of patients undergoing transurethral procedures should be effective against anaerobic bacteria.

Technique for the culture and direct sensitivity testing or large numbers of urine specimens.

A technique was required for batch culturing and direct sensitivity testing of urine specimens to reduce increasing demands on laboratory services. A comparison was made between the filter paper strip method and multipoint inoculator technique using a Denley inoculator (Denley Ltd, Billingshurst, Sussex). The results showed that multipoint inoculation was a rapid, economical, and reliable method for the handling of large numbers of urine samples.

The use of a hydrophobic resin as a product reservoir in steroid transformations.

Particles of the hydrophobic resin polydimethylsiloxane were found to preferentially accumulate steriods on the basis of their hydrophobicity. Thus, the resin selectively sorped the steroid products resulting from the transformation of diosgenin by Nocardia rhodochrous, with the result that higher yields of the later biotransformation product, 1-dehydrodiosgenone, and lower yields of the first product, diosgenone, were obtained than in the absence of resin. Furthermore, steroids accumulated by the resin were available for further biotransformation, so that a two-step reaction forming androstenes from a crude extract of furostanol glycosides (obtained from fenugreek seed) could be carried out. The first step involves deglycosylation and is catalyzed by Fusarium solani. In the presence of resin the water-insoluble diosgenin product becomes sorped to the resin and can be easily transferred to a second fermentation in which diosgenone, 1-dehydrodiosgenone, and androstenes were formed by Mycobacterium phlei. These compounds were completely accumulated by the resin at the end of the fermentation. This procedure is logistically more convenient than the conventional chemical process and illustrates the potential of biotechnological processes in which simultaneous reaction, product isolation, and product purification occur.

The extraction and mechanism of a novel isomaltulose-synthesizing enzyme from Erwinia rhapontici.

The single enzyme that mediates the bioconversion is demonstrated to be located in the cells' periplasmic space, a site that facilitates its use as an industrial biocatalyst, and to be a previously undescribed hexosyltransferase with four novel features. The enzyme is sucrose-specific, and has an intramolecular mechanism in which both glucose and fructose residues appear to be enzyme-bound. Thirdly, it is reaction-non-selective, forming simultaneously isomaltulose and a second hitherto uncharacterized alpha-(1----1)-linked disaccharide (trehalulose), by hydrolysis of sucrose followed by reaction of glucose with the C-6 and C-1 positions of the fructofuranose respectively. Finally, on extended incubation an unusual recycling mechanism caused the concentration of isomaltulose, the kinetically preferred product, to reach a transient maximum concentration and then fall, and the concentration of trehalulose, the thermodynamically favoured product, to rise slowly.

The characterization and interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous.

The physical properties and the methods used for interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous by treatment with Triton X-100, trypsin or buffer alone provide evidence that these forms differ chiefly in the possession or absence of a hydrophobic anchor region connected by a trypsin-sensitive region. The hydrophobic domain normally integrates the enzyme into the cell membrane and confers amphipathic properties on the solubilized enzyme, causing adsorption to hydrophobic resins, aggregation when detergent is removed and formation of mixed micelles with detergent and cholesterol resulting in surface-dilution kinetic behaviour and activation by relatively high concentrations of water-miscible solvents. By contrast, only the enzymic fragment is extracted with trypsin and it behaves as a conventional soluble enzyme and does not aggregate or interact with hydrophobic resins, detergents or water-miscible solvents. As no phospholipid could be detected in the enzyme extracts, the detergent appears to act as a substitute for the cell-membrane lipids that would normally interact with the hydrophobic region. This cholesterol oxidase is an example of a prokaryotic enzyme possessing two closely associated catalytic functions, dehydrogenase and isomerase activities, and an anchoring function.

A benign deficiency of typeB beta-galactosidase in human liver.

The type A or 'acid' and type B or 'neutral' beta-galactosidase activities have been measured in post-mortem liver samples from individuals dying of non-genetic diseases and patients dying of ganglioside storage disease other than GM1 gangliosidosis. The type A activities fell within the established normal range in all samples. The type B activities showed a biomodal distribution suggesting the occurrence of two distinct populations of human individuals. The greater proportion had activities within the range 11.67 pkat/mg of protein (+/- 3.33, S.D.), while others had lower activities in the range 0.48 pkat/mg of protein (+/- 0.38, S.D.). No clinical symptoms were associated with the much lower type B beta-galactosidase activities and it appears that this beta-galactosidase deficiency could be found in the original tissues. Methods of screening for type B beta-galactosidase deficiency are described and the significance of this enzyme deficiency is discussed.

The separation and characterization of the methylumbelliferyl beta-galactosidases of human liver.

1. A previously uncharacterized form of human liver acid beta-galactosidase (EC 3.2.1.23), possibly a dimer of molecular weight 160 000, was resolved by gel filtration. It has the same ability to hydrolyse GM1 ganglioside as the two other acid beta-galactosidase forms. 2. The low-molecular-weight forms of acid beta-galactosidase undergo salt-dependent aggregation. 3. The high-molecular-weight component may consist of the low-molecular-weight forms bound to membrane fragments. It can be converted completely into a mixture of these forms. 4. The neutral beta-galactosidase activity can be resolved into two forms by DEAE-cellulose chromatography. They differ in their response to Cl-ions. 5. A new nomenclature is suggested for the six beta-galactosidases so far found in human liver. 6. The enzymic constituents of the beta-galactosidase bands resolved by electrophoresis were re-examined. The A band contains three components. A two-dimensional electrophoretic procedure for resolving the A band is described. 7. The effect of neuraminidase treatment on the behaviour of beta-galactosidases in various separation systems is examined.