Characterization of the metallo-beta-lactamase determinant of Acinetobacter baumannii AC-54/97 reveals the existence of bla(IMP) allelic variants carried by gene cassettes of different phylogeny.
(49/991)
The metallo-beta-lactamase determinant of Acinetobacter baumannii AC-54/97, a clinical isolate from Italy that was previously shown to produce an enzyme related to IMP-1, was isolated by means of a PCR methodology which targets amplification of gene cassette arrays inserted into class 1 integrons. Sequencing revealed that this determinant was an allelic variant (named bla(IMP-2)) of bla(IMP) found in Japanese isolates and that it was divergent from the latter by 12% of its nucleotide sequence, which evidently had been acquired independently. Similar to bla(IMP), bla(IMP-2) was also carried by an integron-borne gene cassette. However, the 59-base element of the bla(IMP-2) cassette was unrelated to those of the bla(IMP) cassettes found in Japanese isolates, indicating a different phylogeny for the gene cassettes carrying the two allelic variants. Expression of the integron-borne bla(IMP-2) gene in Escherichia coli resulted in a significant decrease in susceptibility to a broad array of beta-lactams (ampicillin, carbenicillin, cephalothin, cefoxitin, ceftazidime, cefepime, and carbapenems). The IMP-2 enzyme was purified from an Escherichia coli strain carrying the cloned determinant, and kinetic parameters were determined with several beta-lactam substrates. Compared to IMP-1, the kinetic parameters of IMP-2 were similar overall with some beta-lactam substrates (cefoxitin, ceftazidime, cefepime, and imipenem) but remarkably different with others (ampicillin, carbenicillin, cephaloridine, and meropenem), revealing a functional significance of at least some of the mutations that differentiate the two IMP variants. Present findings suggest that the environmental reservoir of bla(IMP) alleles could be widespread and raise a question about the global risk of their transfer to clinically relevant species. (+info)
In vitro ATP regeneration from polyphosphate and AMP by polyphosphate:AMP phosphotransferase and adenylate kinase from Acinetobacter johnsonii 210A.
(50/991)
In vitro enzyme-based ATP regeneration systems are important for improving yields of ATP-dependent enzymatic reactions for preparative organic synthesis and biocatalysis. Several enzymatic ATP regeneration systems have been described but have some disadvantages. We report here on the use of polyphosphate:AMP phosphotransferase (PPT) from Acinetobacter johnsonii strain 210A in an ATP regeneration system based on the use of polyphosphate (polyP) and AMP as substrates. We have examined the substrate specificity of PPT and demonstrated ATP regeneration from AMP and polyP using firefly luciferase and hexokinase as model ATP-requiring enzymes. PPT catalyzes the reaction polyP(n) + AMP --> ADP + polyP(n-1). The ADP can be converted to ATP by adenylate kinase (AdK). Substrate specificity with nucleoside and 2'-deoxynucleoside monophosphates was examined using partially purified PPT by measuring the formation of nucleoside diphosphates with high-pressure liquid chromatography. AMP and 2'-dAMP were efficiently phosphorylated to ADP and 2'-dADP, respectively. GMP, UMP, CMP, and IMP were not converted to the corresponding diphosphates at significant rates. Sufficient AdK and PPT activity in A. johnsonii 210A cell extract allowed demonstration of polyP-dependent ATP regeneration using a firefly luciferase-based ATP assay. Bioluminescence from the luciferase reaction, which normally decays very rapidly, was sustained in the presence of A. johnsonii 210A cell extract, MgCl(2), polyP(n=35), and AMP. Similar reaction mixtures containing strain 210A cell extract or partially purified PPT, polyP, AMP, glucose, and hexokinase formed glucose 6-phosphate. The results indicate that PPT from A. johnsonii is specific for AMP and 2'-dAMP and catalyzes a key reaction in the cell-free regeneration of ATP from AMP and polyP. The PPT/AdK system provides an alternative to existing enzymatic ATP regeneration systems in which phosphoenolpyruvate and acetylphosphate serve as phosphoryl donors and has the advantage that AMP and polyP are stabile, inexpensive substrates. (+info)
Monitoring precursor 16S rRNAs of Acinetobacter spp. in activated sludge wastewater treatment systems.
(51/991)
Recently, Cangelosi and Brabant used oligonucleotide probes targeting the precursor 16S rRNA of Escherichia coli to demonstrate that the levels of precursor rRNA were more sensitive to changes in growth phase than the levels of total rRNA (G. A. Cangelosi and W. H. Brabant, J. Bacteriol. 179:4457-4463, 1997). In order to measure changes in the levels of precursor rRNA in activated sludge systems, we designed oligonucleotide probes targeting the 3' region of the precursor 16S rRNA of Acinetobacter spp. We used these probes to monitor changes in the level of precursor 16S rRNA during batch growth of Acinetobacter spp. in Luria-Bertani (LB) medium, filtered wastewater, and in lab- and full-scale wastewater treatment systems. Consistent with the previous reports for E. coli, results obtained with membrane hybridizations and fluorescence in situ hybridizations with Acinetobacter calcoaceticus grown in LB medium showed a more substantial and faster increase in precursor 16S rRNA levels compared to the increase in total 16S rRNA levels during exponential growth. Diluting an overnight culture of A. calcoaceticus grown in LB medium with filtered wastewater resulted in a pattern of precursor 16S rRNA levels that appeared to follow diauxic growth. In addition, fluorescence in situ hybridizations with oligonucleotide probes targeting total 16S rRNA and precursor 16S rRNA showed that individual cells of A. calcoaceticus expressed highly variable levels of precursor 16S rRNA when adapting from LB medium to filtered sewage. Precursor 16S rRNA levels of Acinetobacter spp. transiently increased when activated sludge was mixed with influent wastewater in lab- and full-scale wastewater treatment systems. These results suggest that Acinetobacter spp. experience a change in growth activity within wastewater treatment systems. (+info)
The 1.8 A crystal structure of catechol 1,2-dioxygenase reveals a novel hydrophobic helical zipper as a subunit linker.
(52/991)
BACKGROUND: Intradiol dioxygenases catalyze the critical ring-cleavage step in the conversion of catecholate derivatives to citric acid cycle intermediates. Catechol 1,2-dioxygenases (1, 2-CTDs) have a rudimentary design structure - a homodimer with one catalytic non-heme ferric ion per monomer, that is (alphaFe(3+))(2). This is in contrast to the archetypical intradiol dioxygenase protocatechuate 3,4-dioxygenase (3,4-PCD), which forms more diverse oligomers, such as (alphabetaFe(3+))(2-12). RESULTS: The crystal structure of 1,2-CTD from Acinetobacter sp. ADP1 (Ac 1,2-CTD) was solved by single isomorphous replacement and refined to 2.0 A resolution. The structures of the enzyme complexed with catechol and 4-methylcatechol were also determined at resolutions of 1.9 A and 1.8 A, respectively. While the characteristics of the iron ligands are similar, Ac 1,2-CTD differs from 3,4-PCDs in that only one subunit is used to fashion each active-site cavity. In addition, a novel 'helical zipper', consisting of five N-terminal helices from each subunit, forms the molecular dimer axis. Two phospholipids were unexpectedly found to bind within an 8 x 35 A hydrophobic tunnel along this axis. CONCLUSIONS: The helical zipper domain of Ac 1, 2-CTD has no equivalent in other proteins of known structure. Sequence analysis suggests the domain is a common motif in all members of the 1,2-CTD family. Complexes with catechol and 4-methylcatechol are the highest resolution complex structures to date of an intradiol dioxygenase. Furthermore, they confirm several observations seen in 3,4-PCDs, including ligand displacement upon binding exogenous ligands. The structures presented here are the first of a new family of intradiol dioxygenases. (+info)
An OmpA-like protein from Acinetobacter spp. stimulates gastrin and interleukin-8 promoters.
(53/991)
Bacterial overgrowth in the stomach may occur under conditions of diminished or absent acid secretion. Under these conditions, secretion of the hormone gastrin is elevated. Alternatively, bacterial factors may directly stimulate gastrin. Consistent with this hypothesis, we found that mice colonized for 2 months with a mixed bacterial culture of opportunistic pathogens showed an increase in serum gastrin. To examine regulation of gene expression by bacterial proteins, stable transformants of AGS cells expressing gastrin or interleukin-8 (IL-8) promoters were cocultured with live organisms. Both whole-cell sonicates and a heat-stable fraction were also coincubated with the cells. A level of 10(8) organisms per ml stimulated both the gastrin and IL-8 promoters. Heat-stable proteins prepared from these bacterial sonicates stimulated the promoter significantly more than the live organism or unheated sonicates. A 38-kDa heat-stable protein stimulating the gastrin and IL-8 promoters was cloned and found to be an OmpA-related protein. Immunoblotting using antibody to the OmpA-like protein identified an Acinetobacter sp. as the bacterial species that expressed this protein and colonized the mouse stomach. Moreover, reintubation of mice with a pure culture of the Acinetobacter sp. caused gastritis. We conclude that bacterial colonization of the stomach may increase serum gastrin levels in part through the ability of the bacteria to produce OmpA-like proteins that directly stimulate gastrin and IL-8 gene expression. These results implicate OmpA-secreting bacteria in the activation of gastrin gene expression and raise the possibility that a variety of organisms may contribute to the increase in serum gastrin and subsequent epithelial cell proliferation in the hypochlorhydric stomach. (+info)
OXA-24, a novel class D beta-lactamase with carbapenemase activity in an Acinetobacter baumannii clinical strain.
(54/991)
Acinetobacter baumannii RYC 52763/97, a clinical isolate involved in a prolonged nosocomial outbreak at our hospital, was resistant to all beta-lactams tested, including imipenem and meropenem, which had MICs of 128 and 256 microg/ml, respectively. This strain synthesized three beta-lactamases: a plasmid-mediated TEM-1 beta-lactamase (pI 5.4), an AmpC-type chromosomal cephalosporinase (pI 9.4), and a novel, presumptively chromosomally mediated OXA-related enzyme (pI 9.0) named OXA-24. After cloning and sequencing, the deduced amino acid sequence of the OXA-24 beta-lactamase showed 40% homology with the OXA-10 (PSE-2) and OXA-7 beta-lactamases, 39% homology with the OXA-11 and OXA-5 enzymes, and 33% homology with the LCR-1 beta-lactamase. The amino acid sequence of the OXA-24 beta-lactamase contained the STFK motif found in serine beta-lactamases, but the typical class D triad KTG was replaced by KSG and the motif YGN was replaced by FGN. The OXA-24 beta-lactamase hydrolyzed benzylpenicillin and cephaloridine but lacked activity against oxacillin, cloxacillin, and methicillin. The enzymatic activity was inhibited by chloride ions and by tazobactam (50% inhibitory concentration [IC(50)], 0.5 microM), sulbactam (IC(50), 40 microM), and clavulanic acid (IC(50), 50 microM). Carbapenem MICs for an Escherichia coli transformant (pBMB-1) expressing the cloned OXA-24 enzyme had a fourfold increase. Relative V(max)/K(m) values of 13 and 6 were obtained with imipenem and meropenem, respectively, and a positive microbiological assay result with imipenem was obtained with a purified enzymatic extract of this transformant strain. Therefore, we consider this new beta-lactamase to be involved in the carbapenem resistance of A. baumannii RYC 52763/97. (+info)
Bactericidal and bacteriostatic activity of gemifloxacin against Acinetobacter spp. in vitro.
(55/991)
This study compared the in vitro bacteriostatic activity of gemifloxacin (SB-265805) and a panel of test antimicrobial agents against 100 clinical isolates of Acinetobacter spp. (47 Acinetobacter baumannii, 18 Acinetobacter anitratus, 18 Acinetobacter lwoffii, 13 Acinetobacter calcoaceticus and four other Acinetobacter spp.). Gemifloxacin (MIC(50/90) 0.06/16 mg/L) was more than eight-fold more potent than ciprofloxacin (0.5/>128 mg/L), two- to eight-fold more potent than grepafloxacin, moxifloxacin, levofloxacin, ofloxacin and gatifloxacin, and of similar potency to trovafloxacin and sparfloxacin. Cross-resistance was seen only within the quinolone group and did not extend to non-quinolone antimicrobials. The bactericidal activities of gemifloxacin and the six comparator quinolones were investigated by dose-response and time-kill studies against A. baumannii ATCC 19606 at their optimum bactericidal concentration (OBC) and at 4 x MIC. At the OBC there was no significant difference between the quinolones, but at 4 x MIC gemifloxacin showed superior activity, reducing the viable count by almost 2 log(10) in 30 min compared with a 1 log(10) reduction seen with the other drugs. This enhanced killing extended over 24 h, reducing cell numbers by >4 log(10). These data suggest that gemifloxacin has the potential to be of therapeutic value in the treatment of infection by Acinetobacter spp. (+info)
Comparative activities of ciprofloxacin, clinafloxacin, gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, and trovafloxacin against epidemiologically defined Acinetobacter baumannii strains.
(56/991)
In vitro activities of seven fluoroquinolones against 140 clinical Acinetobacter baumannii isolates representing 138 different strain types were determined. The rank order of activity was clinafloxacin > gatifloxacin > levofloxacin > trovafloxacin > gemifloxacin = moxifloxacin > ciprofloxacin. The 31 outbreak-related A. baumannii strains were significantly more resistant than were 109 sporadic strains. (+info)