Glycerol dehydrogenase (EC 1.1.1.6) and dihydroxyacetone kinase (EC 2.7.1.29) were purified from Citrobacter freundii. The dehydrogenase is a hexamer of a polypeptide of 43,000 Da. The enzyme exhibited a rather broad substrate specificity, but glycerol was the preferred substrate in the physiological direction. The apparent K m s of the enzyme for glycerol and NAD ؉ were 1.27 mM and 57 M, respectively. The kinase is a dimer of a polypeptide of 57,000 Da. The enzyme was highly specific for the substrates dihydroxyacetone and ATP; the apparent K m s were 30 and 70 M, respectively. The DNA region which contained the genes encoding glycerol dehydrogenase (dhaD) and dihydroxyacetone kinase (dhaK) was cloned and sequenced. Both genes were identified by N-terminal sequence comparison. The deduced dhaD gene product (365 amino acids) exhibited high degrees of homology to glycerol dehydrogenases from other organisms and less homology to type III alcohol dehydrogenases, whereas the dhaK gene product (552 amino acids) revealed no significant homology to any other protein in the databases. A large gene (dhaR) of 1,929 bp was found downstream from dhaD. The deduced gene product (641 amino acids) showed significant similarities to members of the 54 bacterial enhancer-binding protein family.Microorganisms such as Citrobacter freundii and Klebsiella pneumoniae are able to grow anaerobically on glycerol as the sole carbon and energy source (19). In the absence of an external oxidant, glycerol is fermented by a dismutation process involving two pathways, one serving for glycerol oxidation and the other for the consumption of the reducing equivalents generated. The oxidation of glycerol is catalyzed by NAD ϩ -linked glycerol dehydrogenase, which converts the substrate to dihydroxyacetone (DHA). This product is then phosphorylated by DHA kinase and funneled to glycolysis (15). Generation of NAD ϩ is achieved by the sequential action of coenzyme B 12 -dependent glycerol dehydratase and NADH-linked 1,3-propanediol dehydrogenase (15). Glycerol is first converted to 3-hydroxypropionaldehyde, which then is reduced to 1,3-propanediol, accounting for about 50 to 66% of the glycerol consumed. The four key enzymes of this pathway are encoded by the dha regulon, the expression of which is induced when DHA or glycerol is present (15, 34). The entire dha regulon was cloned from C. freundii (10) and K. pneumoniae (48), but molecular data are available only for C. freundii.Recently we have subcloned, sequenced, and overexpressed the gene encoding 1,3-propanediol dehydrogenase (9). In this report, we describe the purification of glycerol dehydrogenase (EC 1.1.1.6) and DHA kinase (EC 2.7.1.29) from C. freundii and the cloning, identification, and organization of the corresponding genes. MATERIALS AND METHODSMaterials. Q-Sepharose Fast Flow, Reactive Red, and Blue Sepharose CL-6B were obtained from Pharmacia LKB GmbH (Freiburg, Germany), and hydroxyapatite was from Sigma Chemie (Deisenhofen, Germany). Tris, EDTA, and sodium dodecyl sulfate were from...

This study evaluates the ability of the new fluoroquinolone trovafloxacin to attenuate the inflammatory burst known to occur after initiation of antibiotic treatment in pneumococcal meningitis. After exposure to trovafloxacin or ceftriaxone for 3 h in vitro, Streptococcus pneumoniae was injected intracisternally (i.c.) into rabbits every 3 h over 9 h (n = 6 for each antibiotic). Ceftriaxone-treated S. pneumoniae induced consistently higher CSF leucocyte counts (median 2568/microL versus 543/microL at 6 h; P = 0.03; 4560/microL versus 2207/microL at 18 h; P = 0.03) than trovafloxacin-treated bacteria. Meningitis induced in rabbits by i.c. injection of live S. pneumoniae was treated with equal doses of trovafloxacin or ceftriaxone i.v. (ten per group). The bactericidal rates of both antibacterial agents in CSF were almost identical. In comparison with ceftriaxone, trovafloxacin resulted in lower tumour necrosis factor (TNF) and interleukin 1beta (IL-1beta) CSF levels 2 h after the initiation of treatment (TNF levels, median 26 U/mL versus 141 U/mL; P = 0.02; IL-1beta levels 455 pg/mL versus 1399 pg/mL; P = 0.02). Twelve hours after initiation of therapy, however, TNF and IL-1beta were higher in trovafloxacin-treated animals (TNF, 61 U/mL versus 7 U/mL; P = 0.001; IL-1beta, 4320 pg/mL versus 427 pg/mL; P = 0.006). The increase in CSF lactate was less during trovafloxacin therapy than with ceftriaxone (median: 2.0 mmol/L versus 4.0 mmol/L; P = 0.03). In conclusion, S. pneumoniae treated in vitro with trovafloxacin induced less CSF leucocytosis than ceftriaxone-treated S. pneumoniae. After i.c. inoculation of live S. pneumoniae, trovafloxacin therapy delayed, but did not inhibit, the release of the proinflammatory cytokines TNF and IL-1beta, probably by slowing the liberation of bacterial cell wall components into the subarachnoid space.

Rifabutin is a lipophilic antibacterial with high in vitro activity against many pathogens involved in bacterial meningitis including pneumococci. Resistance to β-lactam antibiotics in pneumococci is not associated with a decreased sensitivity to rifabutin (30 strains from Germany with intermediate penicillin resistance; MIC range of penicillin: 0.125-1 mg/l, MIC of rifabutin: < 0.008-0.015 mg/l). Rifabutin at doses of 0.625, 1.25, 2.5, 5 and 10 mg/kg/h i.v. was investigated in a rabbit model of meningitis using a Streptococcus pneumoniae type 3 (MIC/MBC of rifabutin: 0.015/ 0.06 mg/l). The bacterial density in CSF at the onset of treatment was 7.3 ± 0.6 log CFU/ml (mean ± SD). Rifabutin decreased bacterial CSF titers in a dose-dependent manner [δlog CFU/ml/h (slope of the regression line log CFU/ml vs. time) at a dose of 0.625 mg/kg/h: -0.16 ± 0.06 (n = 3), at 1.25 mg/kg/h: -0.20 ± 0.12 (n = 4), at 2.5 mg/kg/h: -0.24 ± 0.04 (n = 4), at 5 mg/kg/h: -0.31 ± 0.10 (n = 8), and at 10 mg/kg/h: -0.29 ± 0.10 (n = 5)]. At high doses rifabutin was as active as ceftriaxone at 10 mg/kg/h (δlog CFU/ml/h: -0.29 ± 0.10, n = 10). Two and 5 h after initiation of therapy, CSF TNF-α activities were lower with rifabutin 5 mg/kg/h than with ceftriaxone (medians 2 vs. 141 U/ml, p = 0.005 at 2 h; median 51 vs. 120 U/ml 5 h after initiation of therapy, p = 0.04). This did not result, however, in a decrease of indicators of neuronal damage. In conclusion, intravenous rifabutin was bactericidal in experimental pneumococcal meningitis. Provided that a well-tolerated i.v. formulation will be available it may qualify as a reserve antibiotic for pneumococcal meningitis, in particular when strains with a reduced sensitivity to β-lactam antibiotics are the causative pathogens.

The release of lipoteichoic acid (LTA) and teichoic acid (TA) from a Streptococcus pneumoniae type 3 strain during exposure to ceftriaxone, meropenem, rifampin, rifabutin, quinupristin-dalfopristin, and trovafloxacin in tryptic soy broth was monitored by a newly developed enzyme-linked immunosorbent assay. At a concentration of 10 μg/ml, a rapid and intense release of LTA and TA occurred during exposure to ceftriaxone (3,248 ± 1,688 ng/ml at 3 h and 3,827 ± 2,133 ng/ml at 12 h) and meropenem (2,464 ± 1,081 ng/ml at 3 h and 2,900 ± 1,364 ng/ml at 12 h). Three hours after exposure to rifampin, rifabutin, quinupristin-dalfopristin, and trovafloxacin, mean LTA and TA concentrations of less than 460 ng/ml were observed (for each group,P < 0.01 versus the concentrations after exposure to ceftriaxone). After 12 h of treatment, the LTA and TA concentrations were 463 ± 126 ng/ml after exposure to rifampin, 669 ± 303 ng/ml after exposure to rifabutin, and 1,236 ± 772 ng/ml after exposure to quinupristin-dalfopristin (for each group,P < 0.05 versus the concentrations after exposure to ceftriaxone) and 1,745 ± 1,185 ng/ml after exposure to trovafloxacin (P = 0.12 versus the concentration after exposure to ceftriaxone). At 10 μg/ml, bactericidal antibacterial agents that do not primarily affect cell wall synthesis reduced the amount of LTA and TA released during their cidal action againstS. pneumoniae in comparison with the amount released after exposure to β-lactams. Larger quantities of LTA and TA were released after treatment with low concentrations (1× the MIC and 1× the minimum bactericidal concentration) than after no treatment for all antibacterial agents except the rifamycins. This does not support the concept of using a low first antibiotic dose to prevent the release of proinflammatory cell wall components.

The inflammatory response following initiation of antibiotic therapy and parameters of neuronal damage were compared during intravenous treatment with quinupristin/dalfopristin (100 mg/kg as either a short or a continuous infusion) and ceftriaxone (10 mg/kg/h) in a rabbit model of Streptococcus pneumoniae meningitis. With both modes of administration, quinupristin/dalfopristin was less bactericidal than ceftriaxone. However, the concentration of proinflammatory cell wall components (lipoteichoic acid (LTA) and teichoic acid (TA)) and the activity of tumour necrosis factor (TNF) in cerebrospinal fluid (CSF) were significantly lower in the two quinupristin/dalfopristin groups than in ceftriaxone-treated rabbits. The median LTA/TA concentrations (25th/75th percentiles) were as follows: (i) 14 h after infection: 133 (72/155) ng/mL for continuous infusion of quinupristin/dalfopristin and 193 (91/308) ng/mL for short duration infusion, compared with 455 (274/2042) ng/mL for ceftriaxone (P = 0.002 and 0.02 respectively); (ii) 17 h after infection: 116 (60/368) ng/mL for continuous infusion of quinupristin/dalfopristin and 117 (41/247) ng/mL for short duration infusion, compared with 694 (156/2173) ng/mL for ceftriaxone (P = 0.04 and 0.03 respectively). Fourteen hours after infection the median TNF activity (25th/75th percentiles) was 0.2 (0.1/1.9) U/mL for continuous infusion of quinupristin/dalfopristin and 0.1 (0.01/3.5) U/mL for short duration infusion, compared with 30 (4.6/180) U/mL for ceftriaxone (P = 0.02 for each comparison); 17 h after infection the TNF activity was 2.8 (0.2/11) U/mL (continuous infusion of quinupristin/dalfopristin) and 0.1 (0.04/6.1) U/mL (short duration infusion), compared with 48.6 (18/169) U/mL for ceftriaxone (P = 0.002 and 0.001). The concentration of neuron-specific enolase (NSE) 24 h after infection was significantly lower in animals treated with quinupristin/dalfopristin: 4.6 (3.3/5.7) microg/L (continuous infusion) and 3.6 (2.9/4.7) microg/L (short duration infusion) than in those treated with ceftriaxone (17.7 (8.8/78.2) microg/L) (P = 0.03 and 0.009 respectively). In conclusion, antibiotic treatment with quinupristin/dalfopristin attenuated the inflammatory response within the subarachnoid space after initiation of antibiotic therapy. The concentration of NSE in the CSF, taken as a measure of neuronal damage, was lower in quinupristin/dalfopristin-treated rabbits than in ceftriaxone-treated rabbits.