Antibiotic de-escalation in bacteremic urinary tract infections: potential opportunities and effect on outcome.

PURPOSE: The aim of this study was to examine the safety and efficacy of antibiotic de-escalation in patients admitted with bacteremic urinary tract infection (UTI). METHODS: A retrospective chart review of patients admitted to a community-hospital in West Texas with bacteremic UTI during the year 2008. Antibiotic de-escalation was defined as changing the intravenous empiric antibiotic regimen to a culture-directed single agent, given intravenously or orally, with a narrower spectrum than the original empiric regimen. RESULTS: Ninety-seven patients were admitted with bacteremic UTI. Thirty-two patients were not eligible for de-escalation. Among the 65 patients who were eligible for de-escalation, the treating physicians failed to de-escalate antibiotics in 31 cases (47.7%). Fluoroquinolones' resistance, bacteria other than Escherichia coli and discharge to long-term care facilities predicted failure to de-escalate antibiotics. On multivariate analysis, discharge to long-term care facility was the only risk factor that predicted failure to de-escalate antibiotics. The difference between mean hospital length of stay and mortality between the above two groups was not statistically significant. CONCLUSION: Antibiotic de-escalation is under-recognized and sporadically practiced. In patients admitted with bacteremic UTI, empiric antibiotic regimen can be changed to a culture-directed single antibiotic without an increase in hospital length of stay or patients' mortality.

TraI, a LuxI homologue, is responsible for production of conjugation factor, the Ti plasmid N-acylhomoserine lactone autoinducer.

Conjugal transfer of the nopaline-type Agrobacterium Ti plasmid pTiC58 is regulated by a transcriptional activator, TraR, and a diffusible signal molecule, conjugation factor (CF). CF is a member of a family of substituted homoserine lactones (HSLs) that act as coinducers for regulating gene expression in diverse Gram-negative bacteria by a mechanism called autoinduction. In Vibrio fischeri HSL production is conferred by the luxI gene. Homologues of this gene are responsible for HSL production by other Gram-negative bacteria. A gene that we call traI, conferring production of material with CF activity, was localized to a 1-kb region at the upstream end of tra3 of pTiC58. Spectroscopy showed that the activity was authentic CF. Sequence analysis showed that traI could encode a protein of 211 amino acids, TraI, that is related to the proteins responsible for HSL production by other bacteria. A second, partial open reading frame immediately downstream of traI could encode a protein related to TrbB of plasmid RP4, which is required for conjugal transfer. Transcription of traI and of the downstream tra3 genes requires TraR and CF and initiates from the traI promoter. The results show that traI is responsible for CF production, that it is the first gene of the tra3 operon, and that expression of this operon is regulated by autoinduction.

Characterization of genes for synthesis and catabolism of a new rhizopine induced in nodules by Rhizobium meliloti Rm220-3: extension of the rhizopine concept.

Rhizopines are selective growth substrates synthesized in nodules only by strains of rhizobia capable of their catabolism. We report the isolation and study of genes for the synthesis and catabolism of a new rhizopine, scyllo-inosamine (sIa), from alfalfa nodules induced by Rhizobium meliloti Rm220-3. This compound is similar in structure to the previously described rhizopine 3-O-methyl-scyllo-inosamine from R. meliloti L5-30 (P.J. Murphy, N. Heycke, Z. Banfalvi, M.E. Tate, F.J. de Bruijn, A. Kondorosi, J. Tempé, and J. Schell, Proc. Natl. Acad. Sci. USA 84:493-497, 1987). The synthesis (mos) and catabolism (moc) genes for the Rm220-3 rhizopine are closely linked and located on the nod-nif Sym plasmid. The mos genes are directly controlled by the NifA/NtrA regulatory system. A comparison of the sequence of the 5' regions of the two mos loci shows very extensive conservation of sequence as well as strong homology to the nifH coding region. Restriction mapping and hybridization to DNA from the four open reading frames (ORFs) of the L5-30 mos locus indicate the absence of mosA and presence of the other three ORFs (ORF1 and mosB and -C) in Rm220-3. We suggest that the L5-30 mosA gene product is involved in the conversion of scyllo-inosamine to 3-O-methyl-scyllo-inosamine. Restriction fragment length polymorphism analysis of the moc regions of both strains shows that they are very similar. Regulation studies indicate that the moc region is not controlled by the common regulatory gene nifA, ntrA, and ntrC. We discuss the striking similarities in gene structure, location, and regulation between these two rhizopine loci in relation to the rhizopine concept.

Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones.

Conjugal opines secreted by crown gall tumours induce strains of Agrobacterium tumefaciens that are donors of Ti plasmids to produce a diffusible conjugation factor. This enhances the conjugal transfer efficiency of the Ti plasmid in other strains of A. tumefaciens. This factor behaves as a secondary messenger, transmitting the environmental information to tra genes. Here we report the use of spectrometry to show that this factor is identical to synthetic N-(beta-oxo-octan-1-oyl)-L-homoserine lactone and confirm that the synthetic compound is biologically active. N-(Hexan-1-oyl)-L-homoserine lactone has also been detected. A closely related molecule, N-(beta-oxo-hexan-1-oyl)-L-homoserine lactone, autoinduces bioluminescence in the distantly related bacterium, Vibrio fischeri. N-Acyl-homoserine lactones thus seem to be conserved molecules in which the length and nature of the lipophilic acyl chain determines the biological function to be regulated. Mutants that do not produce the factor fail to conjugate unless supplied with it in the induction medium (our unpublished data). These data indicate that the conjugation factor is an autoinducer and a key signal molecule in the conjugation system of A. tumefaciens. It is, to our knowledge, the first example of a second messenger molecule in a bacterial conjugation system.

Structure of the pseudomonad fungal antibiotic phenazine-1-carboxylic acid.

C13H8N2O2, Mr = 224.2, monoclinic, Cc, a = 3.955 (1), b = 19.278 (4), c = 13.468 (1) A, beta = 98.90 (2) degrees, V = 1015 (2) A3, Z = 4, D chi = 1.468 Mg m-3, lambda (Mo K alpha) = 0.7107 A, mu = 0.061 mm-1, F(000) = 464, T = 293 (2) K, R = 0.047 for 571 observed reflections. The crystal-structure determination of the title compound, a phenazine antibiotic from Pseudomonas fluorescens 2-79 (NRRL B-15132), confirms its structure as phenazine-1-carboxylic acid. The molecular packing is described by discrete stacks of molecules parallel to the a axis with the distance between the essentially planar molecules being ca 3.96 A; there are no significant intermolecular contacts in the lattice.

Opine utilization by Agrobacterium spp.: octopine-type Ti plasmids encode two pathways for mannopinic acid degradation.

Octopine-type strains of Agrobacterium tumefaciens degrade the opine mannopinic acid through a specific pathway which involves cleavage of the molecule at the C--N bond between the amino acid and the sugar moieties. Mannose was identified as a product of the reaction. This pathway was inducible by mannopinic and agropinic acids, but not by mannopine or agropine, the two other mannityl opines. The transport system for this pathway appeared to be specific for mannopinic acid. A second, nonspecific pathway for mannopinic acid degradation was also identified. This involved some of the catabolic functions associated with the metabolism of mannopine and agropine. This second pathway was inducible by mannopine and agropine but not by mannopinic or agropinic acids. The transport system for this pathway appeared to have a broad specificity. Transposon Tn5 insertion mutants affected in the specific catabolic pathway were isolated and analyzed. These mutants continued to catabolize mannopine and agropine. Both mapped to a region of the Ti plasmid previously shown to be associated with the catabolism of mannopinic acid. Restriction enzyme analysis of the Ti plasmid from strain 89.10, an octopine strain that is naturally unable to utilize mannopinic acid, showed a deletion in this same region encoding the specific mannopinic acid degradation pathway. Analysis of recombinant clones showed that the second, nonspecific pathway was encoded in a region of the Ti plasmid associated with mannopine and agropine catabolism. This region shared no structural overlap with the segment of the plasmid encoding the specific mannopinic acid degradative pathway.

Revised structure for the phenazine antibiotic from Pseudomonas fluorescens 2-79 (NRRL B-15132).

A phenazine antibiotic (mp, 243 to 244 degrees C), isolated in a yield of 134 micrograms/ml from cultures of Pseudomonas fluorescens 2-79 (NRRL B-15132), was indistinguishable in all of its measured physicochemical (melting point, UV and infrared spectra, and gas chromatography-mass spectrometry data) and biological properties from synthetic phenazine-1-carboxylic acid. Gurusiddaiah et al. (S. Gurusiddaiah, D. M. Weller, A. Sarkar, and R. J. Cook, Antimicrob. Agents Chemother. 29:488-495, 1986) attributed a dimeric phenazine structure to an antibiotic with demonstrably similar properties obtained from the same bacterial strain. Direct comparison of the physicochemical properties of the authentic antibiotic obtained from D. M. Weller with synthetic phenazine-1-carboxylic acid and with the natural product from the present study established that all three samples were indistinguishable within the experimental error of each method. No evidence to support the existence of a biologically active dimeric species was obtained. Phenazine-1-carboxylic acid has a pKa of 4.24 +/- 0.01 (25 degrees C; I = 0.09), and its carboxylate anion shows no detectable antimicrobial activity compared with the active uncharged carboxylic acid species. These data suggest that phenazine-1-carboxylic acid is probably not an effective biological control agent for phytopathogens in environments with a pH greater than 7.

Genes for the catabolism and synthesis of an opine-like compound in Rhizobium meliloti are closely linked and on the Sym plasmid.

In alfalfa nodules induced by Rhizobium meliloti strain L5-30 the compound L-3-O-methyl-scyllo-inosamine (3-O-MSI) is synthesized. This compound is also catabolized specifically by this strain. Its biological properties are therefore similar to the Agrobacterium opines. To answer the question whether opine-like compounds ("Rhizopines") play a role in a plant symbiotic interaction, we isolated the genes for the catabolism of 3-O-MSI (moc genes) and for the induction of its synthesis in the nodule [mos gene(s)]. moc and mos genes were shown to be closely linked and located on the Sym plasmid of L5-30, suggesting that they have co-evolved and may be important in symbiosis. These genes have been cloned into a broad host-range vector that can be mobilized into other R. meliloti strains where they are expressed. The location of the mos genes in the bacteria extends the opine concept, initially developed for a plant pathological interaction, to a symbiotic one.

Virulence properties of strains of agrobacterium on the apical and Basal surfaces of carrot root discs.

Most pathogenic strains of Agrobacterium are able to induce crown gall or hairy root on both the apical surface (facing the root tip) and the basal surface (facing the shoot) of carrot (Daucus carota L.) root discs. Tumorigenic strains carrying mutations in the shoot inhibition region of the T-DNA (TL-DNA genes 1 and 2) are markedly attenuated on the basal surface but remain virulent on the apical surface. Coinoculation of two attenuated tumorigenic strains, with mutations in gene 1 and gene 2, respectively, resulted in restoration of virulence on the basal surface. Wild type hairy root-inducing strains can be divided into two groups: those that are virulent on both apical and basal surfaces and those that are virulent only on the apical surface. alpha-Naphthalene acetic acid stimulated virulence of hairy root strain TR7, belonging to the latter group, on the basal surface. Attenuated virulence on the basal surface can be explained in terms of an auxin deficiency in the basal tissues and unidirectional auxin transport to the apical surface.

Agrocinopine A, a tumor-inducing plasmid-coded enzyme product, is a phosphodiester of sucrose and L-arabinose.

Opines are unusual compounds found specifically in plant crown gall tumors. Genes for their synthesis and catabolism reside in agrobacteria as tumor-inducing (Ti) plasmid DNA. Only a small Ti-plasmid segment (24 kilobase pairs), the T-DNA, is transferred to the plant cell where it commonly codes for enzymes involved in the biosynthesis of nitrogenous opines such as nopaline (N2-(1,3-D-dicarboxypropyl)-L-arginine) as well as the tumor phenotype. Ellis and Murphy, (Ellis, J.G., and Murphy, P.J. (1981) Mol. Gen. Genet. 181, 36-43) reported the existence of the phosphorylated opines, agrocinopines A and B in tumors containing nopaline. Pure agrocinopine A has now been isolated in a yield of 0.05-0.06 g/100 g, fresh weight, from such tumors. Physical, chemical, and biological data establish the structure of agrocinopine A as an unusual non-nitrogenous opine of sucrose and L-arabinose with a phosphodiester linkage from the 2-hydroxyl of the arabinose to the 4-hydroxyl of the fructose moiety in sucrose. Agrocinopine B is the corresponding phosphodiester, in which the glucose has been hydrolyzed from the sucrose portion of agrocinopine A. Borohydride reduction of the free L-arabinose anomeric carbon of agrocinopine A, to the corresponding arabinitol derivative eliminates the characteristic inhibition zone enhancement produced by both agrocinopines A and B in the agrocin 84 (a fraudulent adenine nucleotide) bioassay. Because of the limited number of genes in the T-DNA, a generalization is proposed, whereby all opines will be found to comprise two common plant cell constituents linked in an uncommon manner by the minimum number of enzymes.

Agrocins and the biological control of crown gall.

Agrocin 84 is a plasmid-encoded, fraudulent adenine nucleotide antibiotic responsible for the preventative biological control of the plant cancer, crown gall. It has bacteriocin-like selectivity which is dependent on a Ti-plasmid-encoded permease in pathogenic agrobacteria. Other nucleotide agrocins have been described and partially characterized; more may be confidently predicted.

Oxidation of ammonia by Nitrosomonas europaea. Definite 18O-tracer evidence that hydroxylamine formation involves a monooxygenase.

NH2OH, the first intermediate in the oxidation of NH4+ to nitrite by the nitrifying bacterium, Nitrosomonas europaea, was recovered as the oxime of cyclohexanone. 15N, 18O-tracer experiments using highly enriched 15NH4Cl and 18O2 yielded oxime that was correspondingly highly enriched (greater than or equal to 92 atom %) in these isotopes. These results show that the source of NH2OH is largely or entirely NH4+, as opposed to hydrazine, which was added to inhibit the further oxidation of NH2OH to nitrite, and that NH4+ yields NH2OH by way of a monooxygenase reaction involving direct insertion of O from O2. The oxidation of NH4+ and NH2OH must be functionally linked in N. europaea, inasmuch as the reducing equivalents required by the monooxygenase to reduce the second atom of O2 to water can arise only through the concomitant oxidation of NH2OH.

Phosphoglycopeptide, a major constituent of the spermatophoric plasma of the octopus (Octopus dofleini martini).

A phosphoglycopeptide, accounting for approximately 90% of the characteristically high content of acid-soluble organically-bound phosphorus in the octopus spermatophoric plasma (4 mg P/ml), was identified. Electrophoretic and chromatographic purification, followed by chemical and enzymic hydrolysis, yielded D-galactose phosphate as a degradation product. The galactose and peptide moieties of the compound were linked via a phosphoryl rather than a glycosidic linkage but the peptide was devoid of aromatic amino acids.

Determination of ionization constants by paper electrophoresis.

Dimensionless apparent ionization constants of charged low-molecular-weight species may be obtained from paper-electrophoretic data at 20-25 degrees C with buffers (I0.1-0.5) of measured pH (1.5-12.5) containing oxalate ions. Relative mobilities rather than absolute mobilities were measured by using glycerol and m-nitrobenzenesulphonate respectively as standards of zero and unit mobility. Application of the procedure to ionizations of adenine, adenosine, 2'-deoxyadenosine, 3'-deoxyadenosine, 3':5'-cyclic AMP, ADP, ADP-glucose-agrocin 84 and ATP is described.

Substituents at N6 and C-5' control selective uptake and toxicity of the adenine-nucleotide bacteriocin, agrocin 84, in Agrobacteria.

The inhibition of a sensitive strain of Agrobacterium radiobacter by the nucleotide bacteriocin agrocin 84 has been studied. A structure-function study of the agrocin 84 molecule was undertaken. Two agrocin 84 nucleotide fragments lacking either the N6 or 5'-phosphoramidate substituents were used in uptake studies of [32P2]agrocin 84. It was established that the plasmid-controlled, strain-specific uptake of agrocin 84 is determined by the N6-D-glucofuranosyloxyphosphoramidate substituent. This conclusion is further supported by the markedly reduced uptake of the 32P-labelled fragment lacking the N6 substituent. Equilibrium dialysis studies also indicate that the N6 substituent is 'recognised' by a binding protein involved in the uptake of agrocin 84 into sensitive strains. The nucleotide fragment bearing the N6 substituent is a competitive inhibitor for the uptake of agrocin 84 in vivo with a Ki = 1.0 x 10(-7) M and is itself selectively transported into a sensitive strain at a rate comparable with agrocin 84, but unlike agrocin 84 is non-toxic. By contrast, the fragment bearing the 5'-phosphoramidate is taken up by both sensitive and insensitive strains at a barely measurable rate and is toxic to both.

[Preparation and properties of new catabolic substrates for two types of oncogenic plasmids of Agrobacterium tumefaciens]. / Préparation et propriétés de nouveaux substrats cataboliques pour deux types de plasmides oncogènes d'Agrobacterium tumefaciens.

Reduction of the Schiff bases formed between glucose, mannose or galactose with glutamic acid yields products related both structurally and biologically to agropine and to a derivative of agropine present in crown gall tumours. The catabolism of these compounds is coded for by octopine and agropine Ti-plasmids of Agrobacterium tumefaciens.