In Salmonella enterica, there is a great variety of 0 antigens, each consisting of a short oligosaccharide (

A two-step enzymatic synthesis of dTDP-L-rhamnose is developed using enzymes from sonicated extracts of cultures of Escherichia coli K12 strains harboring plasmids containing different parts of the rjb gene cluster of Salmonella enterica LT2. The intermediate dTDP-6-deoxy-~-xylo-4-hexulose was isolated after a 1 -h reaction, using only dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase, followed by protein precipitation and desalting by gel chromatography (yield 89Y0). In a two-step reaction using dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase in the first step, and with NADPH, dTDP-6-deoxy-~-xylo-4-hexulose 3,5-epimerase and NADPH : dTDP-6-deoxy-~-l.vxo-4-hexulose-4-reductase in the second hour of incubation, the dTDP-D-glucose was fully converted to dTDP-L-rhamnose. The hexoses of both products were identified by mass spectroscopy. The molar yield of dTDP-L-rhamnose, after protein precipitation, anion-exchange chromatography and desalting by gel chromatography, was ~W O , corresponding to more than 150 mg, starting from 250 mg of dTDP-D-glucose. When stored lyophilysed under nitrogen, these products were found to be stable for several months. Both dTDP-6-deoxy-~-xylo-4-hexulose and dTDP-L-rhamnose have light absorption maxima at 267 nm, with molar absorption coefficients close to that of dTMP. However, the absorption coefficient of dTDP-6-deoxy-~-.xylo-4-hexulose at the absorption maximum of 320 nm (specific for sugars containing keto groups) was found to be approximately 20% higher than values presented earlier.Furthermore, an HPLC technique is presented for determining the net activity of dTDP-6-deoxy-~-xy/o-4-hexulose 3,5-epimerase and NADPH : dTDP-6-deoxy-~-lyxo-4-hexulose-4-reductase, based on separation of dTDP-6-deoxy-~-xylo-4-hexulose and dTDP-L-rhamnose. The HPLC technique is also suitable for determination of all the nucleotide components involved in the synthesis.The 0 antigen part of the lipopolysaccharide of Salmonella enterica sero-group B strains has a saccharide chain built of site interaction. Therefore, isolated saccharides are important for elucidation of host-defence mechanisms.In recent years advances have been made in the chemical synthesis of saccharides [3]. However, saccharide synthesis is still a relatively complicated procedure, in particular when saccharides larger than the tetrasaccharides shown above are to be synthesized and synthesis is required in large quantities. Such saccharides may be required for the production of synthetic vaccines. S. entericu 0-antigen-specific oligosaccharides have also been produced by enzymatic degradation of the polysaccharide, utilizing bacteriophage glycosidases [4]. So far this procedure has yielded oligosaccharides (monotetramers, ditetramers, tritetramers, and tetramers of a tetrasaccharide) which have terminal sugars different from those of the natural biological repeating unit. We have therefore investigating the feasability of enzymatic synthesis of the S. enterica 0-antigenspecific oligosaccharide using enzymes overproduced by cloned gene...

Earlier studies have reported very high (120 to 2,700 mg/kg) concentrations of norfloxacin in feces after therapeutic doses. MICs for fecal microorganisms are with few exceptions far below these levels. Nevertheless, clinical investigations show that the main part of the aerobic gram-positive and the anaerobic microflora remains unaffected after norfloxacin administration. In Previous studies on the newer quinolones have shown that administration of these agents causes selective ecological disturbances in the gastrointestinal flora (11,20). Several investigations including healthy volunteers as well as different categories of patients on the impact of norfloxacin on the normal gastrointestinal microflora showed uniform results (2, 9, 17, 18). The aerobic gram-negative microorganisms are eliminated or strongly suppressed during administration, while the aerobic gram-positive and the anaerobic microfloras are unaffected. These favorable ecological properties of norfloxacin contribute to a preservation of "colonization resistance" (25) and thus make norfloxacin suitable for long-term prophylactic treatment of urinary tract infections, selective decontamination in immunocompromised patients, and treatment of bacterial intestinal infections.However, very high concentrations of norfloxacin are achieved in feces. Peak fecal concentrations of norfloxacin of 120 to 2,700 mg/kg after therapeutic doses have been reported (5, 9, 18). MICs for both aerobic and anaerobic microorganisms are with few exceptions far below these levels (10,19,22). Thus, despite the high levels of norfloxacin in feces, the main part of the aerobic gram-positive and the anaerobic flora remains unaffected after norfloxacin administration. Different theories have been raised to explain this paradox. Differences in anaerobiosis of the colon environment in vivo compared with the in vitro conditions have been suggested to influence the sensitivity of anaerobic bacteria. An inoculum effect on the activity of norfloxacin has been shown for anaerobic strains (12). At an inoculum of 109 CFU/ml, norfloxacin concentrations greater than 512 ,ug/ ml are required to reduce most gram-positive and gramnegative fecal anaerobes to less than 103 CFU/ml. The third * Corresponding author. theory suggests that there is a binding between norfloxacin and fecal material. Reversible binding to feces has been seen for other antimicrobial agents (13,14). The influence of feces on the antimicrobial activity of norfloxacin has been studied (26), but to our knowledge no study has characterized the binding of quinolones to feces.

ClinicalTrials.gov, NCT02066428 and NCT02074956.