The Universal Method (UM) described here will allow the detection of any bacterial rDNA leading to the identification of that bacterium. The method should allow prompt and accurate identification of bacteria. The principle of the method is simple; when a pure PCR product of the 16S gene is obtained, sequenced, and aligned against bacterial DNA data base, then the bacterium can be identified. Confirmation of identity may follow. In this work, several general 16S primers were designed, mixed and applied successfully against 101 different bacterial isolates. One mixture, the Golden mixture7 (G7) detected all tested isolates (67/67). Other golden mixtures; G11, G10, G12, and G5 were useful as well. The overall sensitivity of the UM was 100% since all 101 isolates were detected yielding intended PCR amplicons. A selected PCR band from each of 40 isolates was sequenced and the bacterium identified to species or genus level using BLAST. The results of the UM were consistent with bacterial identities as validated with other identification methods; cultural, API 20E, API 20NE, or genera and species specific PCR primers. Bacteria identified in the study, covered 34 species distributed among 24 genera. The UM should allow the identification of species, genus, novel species or genera, variations within species, and detection of bacterial DNA in otherwise sterile samples such as blood, cerebrospinal fluid, manufactured products, medical supplies, cosmetics, and other samples. Applicability of the method to identifying members of bacterial communities is discussed. The approach itself can be applied to other taxa such as protists and nematodes.

Aeromonas hydrophila 495A2 excreted two forms of amonabactin, a new phenolate siderophore composed of 2,3-dihydroxybenzoic acid, lysine, glycine, and either tryptophan (amonabactin T) or phenylalanine (amonabactin P). Supplementing cultures with L-tryptophan (0.3 mM) caused exclusive synthesis of amonabactin T, whereas supplements of L-phenylalanine (0.3 to 30 mM) gave predominant production of amonabactin P. The two forms of amonabactin were separately purified by a combination of production and polyamide column chromatographic methods. Both forms were biologically active, stimulating growth in iron-deficient medium of an amonabactin-negative mutant. Of 43 additional siderophore-producing isolates of the Aeromonas species that were tested, 76% (19 of 25) of the A. hydrophila isolates were amonabactin positive, whereas only 19% (3 of 16) of the A. sobria isolates and all (3 of 3) of the A. caviae isolates produced amonabactin, suggesting a predominant synthesis of amonabactin in certain Aeromonas species.Aeromonas hydrophila and related aeromonads are gramnegative, freshwater pathogens of fish and humans. In fish they cause a fatal hemorrhagic septicemia, and in humans they cause wound, soft tissue, and blood infections as well as acute gastroenteritis (2,(5)(6)(7)14 Purification of amonabactin. Amonabactin T and amonabactin P were separately purified from supernatants of A. hydrophila 495A2 after its growth in a low-iron minimal medium composed of the following (per liter): glucose, 5 g; (NH4)2HP04, 1 g; K2HPO4, 4 g; KH2PO4, 2.7 g. To lower metal contamination, the medium was treated with Chelex-100 (Bio-Rad Laboratories, Richmond, Calif.) by previously reported methods (1). After the Chelex-treated medium was filter sterilized, it was supplemented with filter sterilized solutions of high purity sulfate salts (Johnson-Matthey, Inc., Seabrook, N.H.) of magnesium (830 ,uM), manganese (40 ,uM), and iron (0.18 ,uM). were incubated at 30°C with aeration at 8 liters per min in a modified model 43-100 fermentor (The Virtis Co., Gardiner, N.Y.) in which exposed stainless steel components were coated with Teflon. For preparation of amonabactin T (the tryptophan-containing form of amonabactin), the medium was supplemented with 0.3 mM L-tryptophan, which caused exclusive production of amonabactin T. At maximum growth (usually after 12 to 18 h), the catecholate siderophore in the supernatant was readily detected by assay for dihydroxy phenolates (4) and by mixing 1 ml of supernatant with 0.005 ml of 1% ferric chloride, which resulted in a bluepurple color. The cells were removed by centrifuging the culture, and the phenolate(s) was adsorbed to polyamide (11) by passing the supernatant through a 5.5-by 13-cm column of polyamide (Woelm, Universal Adsorbents, Atlanta, Ga.) that had bren previously washed with methanol, acetone, and sufficient water to remove the organic solvents. The column then was washed with water (1.5 liters) and then washed with 500 ml of 100% acetone. The amonabactin T then was eluted with methanol (...

Many isolates of the Aeromonas species produce amonabactin, a phenolate siderophore containing 2,3-dihydroxybenzoic acid (2,3-DHB). An amonabactin biosynthetic gene (amoA) was identified (in a Sau3A1 gene library of Aeromonas hydrophila 495A2 chromosomal DNA) by its complementation of the requirement of Escherichia coli SAB11 for exogenous 2,3-DHB to support siderophore (enterobactin) synthesis. The gene amoA was subcloned as a SalI-HindIII 3.4-kb DNA fragment into pSUP202, and the complete nucleotide sequence of amoA was determined. A putative iron-regulatory sequence resembling the Fur repressor protein-binding site overlapped a possible promoter region. A translational reading frame, beginning with valine and encoding 396 amino acids, was open for 1,188 bp. The C-terminal portion of the deduced amino acid sequence showed 58% identity and 79% similarity with the E. coli EntC protein (isochorismate synthetase), the first enzyme in the E. coli 2,3-DHB biosynthetic pathway, suggesting that amoA probably encodes a step in 2,3-DHB biosynthesis and is the A. hydrophila equivalent of the E. coli entC gene. An isogenic amonabactin-negative mutant, A. hydrophila SB22, was isolated after marker exchange mutagenesis with Tn5-inactivated amoA (amoA::Tn5). The mutant excreted neither 2,3-DHB nor amonabactin, was more sensitive than the wild-type to growth inhibition by iron restriction, and used amonabactin to overcome iron starvation.

Adherence of Pseudomonas aeruginosa to cells of the respiratory tract of patients with cystic fibrosis (CF) appears to be a necessary precondition for colonization and infection. To date no effective anti-adhesive strategy has been devised for preventing P. aeruginosa infection in these vulnerable hosts. The purpose of these studies was to evaluate the potential for preventing adhesion of P. aeruginosa to epithelial cells with dextran. Dextran (3,000-70,000 MW) inhibited adhesion of P. aeruginosa to buccal and A549 pulmonary epithelial cells; the 3,000 MW compound, at 10 mM was most inhibitory. Adhesion was inhibited optimally at pH 7.4 and was independent of charge; dextran and dextran sulfate were equally inhibitory. Dextran was most inhibitory if added to the epithelial cells before the P. aeruginosa; adhesion was reversed only minimally by adding dextran after the bacteria were bound. The inhibitory effect appeared to be nonspecific because other neutral polysaccharides (glycogen and mannan) were also inhibitory, dextran blocked attachment of other respiratory tract pathogens (Staphylococcus aureus, Group A streptococcus, and Haemophilus influenzae), and because dextran did not bind specifically to bacteria or to epithelial cells. Dextran is an inexpensive and nontoxic agent and may be useful in patients with CF to prevent colonization and infection with P. aeruginosa.