Detection and molecular characterization of ß-lactamase genes in clinical isolates of Gram-negative bacteria in Southern Ecuador.

This work performed a phenotypic and genotypic characterization of 79 clinical isolates of Enterobacteriaceae and Pseudomonadaceae collected in hospitals of Southern Ecuadorin 2013. Our results showed a high incidence of ß-lactamases and ESBLs with blaTEM and blaCTX-M as the prevalent genes, respectively. By direct sequencing of PCR amplicons, the different ß-lactamases and variants of the genes were also distinguished. Our results revealed a predominance of TEM-1 ß-lactamase and the presence of different CTX-M variants with a prevalence of CTX-M-15. Two infrequent CTX-M variants in South America were also identified. To the best of our knowledge, this is one of the first studies describing the genetic characteristics of ß-lactamases in Ecuador.

An enzymatic glucose/O2 biofuel cell operating in human blood.

Enzymatic biofuel cells (BFCs) may power implanted medical devices and will rely on the use of glucose and O2 available in human bodily fluids. Other than well-established experiments in aqueous buffer, little work has been performed in whole human blood because it contains numerous inhibiting molecules. Here, we tested our BFCs in 30 anonymized, random and disease-free whole human blood samples. We show that by designing our anodic and cathodic bioelectrocatalysts with osmium based redox polymers and home-made enzymes we could reach a high selectivity and biofunctionnality. After optimization, BFCs generate power densities directly proportional to the glycaemia of human blood and reached a maximum power density of 129µWcm(-2) at 0.38V vs. Ag/AgCl at 8.22mM glucose. This is to our knowledge the highest power density attained so far in human blood and open the way for the powering of integrated medical feedback loops.

Promiscuous metallo-ß-lactamases: MIM-1 and MIM-2 may play an essential role in quorum sensing networks.

MIM-1 and MIM-2 are two recently identified metallo-ß-lactamases (MBLs) from Novosphingobium pentaromativorans and Simiduia agarivorans, respectively. Since these organisms are non-pathogenic we speculated that the biological role(s) of MIM-1 and MIM-2 may not be related to their MBL activity. Although both sequence comparison and homology modeling indicate that these proteins are homologous to well-known MBLs such as AIM-1, the sequence analysis also indicated that MIM-1 and MIM-2 share similarities with N-acyl homoserine lactonases (AHLases) and glyoxalase II (GLX-II). Steady-state kinetic assays using a series of lactone substrates confirm that MIM-1 and MIM-2 are efficient lactonases, with catalytic efficiencies resembling those of well-known AHLases. Interestingly, unlike their MBL activity the AHLase activity of MIM-1 and MIM-2 is not dependent on the metal ion composition with Zn(II), Co(II), Cu(II), Mn(II) and Ca(II) all being able to reconstitute catalytic activity (with Co(II) being the most efficient). However, these enzymes do not turn over S-lactoylglutathione, a substrate characteristic for GLX-II activity. Since lactonase activity is linked to the process of quorum sensing the bifunctional activity of "non-pathogenic" MBLs such as MIM-1 and MIM-2 may provide insight into one possible evolutionary pathway for the emergence of antibiotic resistance.

Isolation and characterization of halophilic bacteria from Urmia Lake in Iran.

Urmia Lake is one of the most permanent hypersaline lakes in the world which is threatened by hypersalinity and serious dryness. In spite of its importance no paper has been published regarding bacterial community of this lake. Accordingly, the present study aimed to investigate the halophilic bacteria in the aforementioned lake. In so doing, thirty seven strains were isolated on six different culture media. The isolated strains were characterized using phenotypic and genotypic methods. Growth of the strains occurred at 2535 degrees C, pH 6-9 and 7 to 20% (w/v) NaCl indicating that most of the isolates were moderately halophiles. Catalase, oxidase and urease activities were found to be positive for the majority of the isolates. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolated bacteria belonged to two major taxa: Gammaproteobacteria (92%, including Salicola [46%], Pseudomonas [13.5%], Marinobacter [ 11%], Idiomarina [11%], and Halomonas [8%]) and Firmicutes (8%, including Bacillus [5%] and Halobacillus [3%]). In addition, a novel bacterium whose 16S rRNA gene sequence showed almost 98% sequence identity with the taxonomically troubled DSM 3050T, Halovibrio denitrificans HGD 3T and Halospina denitrificans HGD 1-3T, each, was isolated. 16S rRNA gene similarity levels along with phenotypic characteristics suggest that some of the isolated strains could be regarded as potential type strain for novel species, on which further studies are recommended.

Prevalence of extended-spectrum beta-lactamases-producing microorganisms in nosocomial patients and molecular characterization of the shv type isolates

The emergence of Extended-Spectrum Beta-Lactamase (ESBL)-producing microorganisms in Brazilian hospitals is a challenge that concerns scientists, clinicians and healthcare institutions due to the serious risk they pose to confined patients. The goal of this study was the detection of ESBL production by clinical strains of Escherichia coli and Klebsiella sp. isolated from pus, urine and blood of patients at Hospital Universitário Santa Maria, Rio Grande Sul, RS, Brazil and the genotyping of the isolates based on bla SHV genes. The ESBL study was carried out using the Combined Disc Method, while Polymerase Chain Reaction (PCR) was used to study the bla SHV genes. Of the 90 tested isolates, 55 (61.1 percent) were identified as ESBL-producing by the combined disk method. The bla SHV genes were found in 67.8 percent of these microorganisms. K. pneumoniae predominated in the samples, presenting the highest frequency of positive results from the combined disk and PCR.

Comparison of bioassays by testing whole soil and their water extract from contaminated sites.

The harmful effects of contaminants on the ecosystems and humans are characterised by their environmental toxicity. The aim of this study was to assess applicability and reliability of several environmental toxicity tests, comparing the result of the whole soils and their water extracts. In the study real contaminated soils were applied from three different inherited contaminated sites of organic and inorganic pollutants. The measured endpoints were the bioluminescence inhibition of Vibrio fischeri (bacterium), the dehydrogenase activity inhibition of Azomonas agilis (bacterium), the reproduction inhibition of Tetrahymena pyriformis (protozoon), and Panagrellus redivivus (nematode), the mortality of Folsomia candida (springtail), the root and shoot elongation inhibition of Sinapis alba (plant: white mustard) and the nitrification activity inhibition of an uncontaminated garden soil used as "test organism". Besides the standardised or widely used methods some new, direct contact ecotoxicity tests have been developed and introduced, which are useful for characterisation of the risk of contaminated soils due to their interactive nature. Soil no. 1 derived from a site polluted with transformer oil (PCB-free); Soil no. 2 originated from a site contaminated with mazout; Soil no. 3 was contaminated with toxic metals (Zn, Cd, Cu, Pb, As). In most cases, the interactive ecotoxicity tests indicated more harmful effect of the contaminated soil than the tests using soil extracts. The direct contact environmental toxicity tests are able to meet the requirements of environmental toxicology: reliability, sensibility, reproducibility, rapidity and low cost.

Extracellular chitinases of fluorescent pseudomonads antifungal to Fusarium oxysporum f. sp. dianthi causing carnation wilt.

Vascular wilt of carnation caused by Fusarium oxysporum f. sp. dianthi (Prill. & Delacr.) W. C. Synder & H.N. Hans inflicts substantial yield and quality loss to the crop. Mycolytic enzymes such as chitinases are antifungal and contribute significantly to the antagonistic activity of fluorescent pseudomonads belonging to plant-growth-promoting rhizobacteria. Fluorescent pseudomonads antagonistic to the vascular wilt pathogen were studied for their ability to grow and produce chitinases on different substrates. Bacterial cells grown on chitin-containing media showed enhanced growth and enzyme production with increased anti-fungal activity against the pathogen. Furthermore, the cell-free bacterial culture filtrate from chitin-containing media also significantly inhibited the mycelial growth. Both the strains and their cell-free culture filtrate from chitin-amended media showed the formation of lytic zones on chitin agar, indicating chitinolytic ability. Extracellular proteins of highly antagonistic bacterial strain were isolated from cell-free extracts of media amended with chitin and fungal cell wall. These cell-free conditioned media contained one to seven polypeptides. Western blot analysis revealed two isoforms of chitinase with molecular masses of 43 and 18.5 kDa. Further plate assay for mycelial growth inhibition showed the 43-kDa protein to be antifungal. The foregoing studies clearly established the significance of chitinases in the antagonism of fluorescent pseudomonads, showing avenues for possible exploitation in carnation wilt management.

Detection and characterisation of catechol 2,3-dioxygenase in an indigenous soil pseudomonad by MALDI-TOF MS using a column separation.

The key enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway (C230) has been purified to homogeneity from a new bacterial strain, which belongs to genus Pseudomonas. The species was growing on phenol as carbon source. The C230 was detected and identified by absorption spectroscopy. The protein was isolated using sucrose density centrifugation and anion exchange chromatography. The purified protein showed a molecular mass of 32 kDa to sodium dodecyl sulfate polyacrylamid gel electrophoresis and an isoelectric point of 5 estimated by analytical isoelectrical focusing. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and peptide mapping was attempted for the identification of the isolated protein and proteins involved in the metabolic pathway.

The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation.

beta-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.

Use of Etests with carbapenems for Gram-negative rods producing beta-lactamases.

The in vitro activity of imipenem and meropenem on strains of Gram-negative rods producing extended-spectrum beta-lactamase (ESBL) and inducible beta-lactamase (IBL) was studied using the Etest. In all, 185 strains from the surgical intensive care units of four different hospitals were looked at over 2 years. Of these, 94 were ESBL producers and 91 were IBL positive. The in vitro sensitivities of imipenem and meropenem were 89.7 and 95.1%, respectively, against all strains. The imipenem and meropenem sensitivities of Klebsiella spp. were 98.4 and 100%, respectively, but imipenem resistance (21.6%) in Pseudomonas aeruginosa strains was higher than that of meropenem (10.8%).

Two energetically disparate folding pathways of alpha-lytic protease share a single transition state.

The Lysobacter enzymogenes alpha-lytic protease (alphaLP) is synthesized with a 166 amino acid pro region (Pro) that catalyzes the folding of the 198 amino acid protease into its native conformation. An extraordinary feature of this system is the very high energy barrier (DeltaG = 30 kcal mol-1) that effectively prevents alphaLP from folding in the absence of Pro (t1/2 = 1800 years). A pair of mutations has been isolated in the protease that completely suppresses the catalytic defect incurred in Pro by truncation of its last three amino acids. These mutations also accelerate the folding of alphaLP in the absence of Pro by 400-fold. An energetic analysis of the two folding reactions indicates that the mutations stabilize the transition states of both the catalyzed and uncatalyzed folding reactions by 3 kcal mol-1. This finding points to a single transition state for these two distinct and energetically disparate folding pathways, and raises the possibility that all alphaLP folding pathways share the same transition state.

Identification of genes unique to Mo-independent nitrogenase systems in diverse diazotrophs.

A number of nitrogen-fixing bacteria were screened using PCR for genes (vnfG and anfG) unique to the V-containing nitrogenase (vnf) and the Fe-only nitrogenase (anf) systems. Products with sequences similar to that of vnfG were obtained from Azotobacter paspali and Azotobacter salinestris genomic DNAs, and products with sequences similar to that of anfG were obtained from Azomonas macrocytogenes, Rhodospirillum rubrum, and Azotobacter paspali DNAs. Phylogenetic analysis of the deduced amino acid sequences of anfG and vnfG genes shows that each gene product forms a distinct cluster. Furthermore, amplification of an internal 839-bp region in anfD and vnfD yielded a product similar to anfD from Heliobacterium gestii and a product similar to vnfD from Azotobacter paspali and Azotobacter salinestris. Phylogenetic analysis of NifD, VnfD, and AnfD amino acid sequences indicates that AnfD and VnfD sequences are more closely related to each other than either is to NifD. The results of this study suggest that Azotobacter salinestris possesses the potential to express the vanadium (V)-containing nitrogenase (nitrogenase 2) and that R. rubrum, Azomonas macrocytogenes, and H. gestii possess the potential to express the Fe-only nitrogenase (nitrogenase 3). Like Azotobacter vinelandii, Azotobacter paspali appears to have the potential to express both the V-containing nitrogenase and the Fe-only nitrogenase.

[Occurrence of extended spectrum beta-lactamases (ESBL) and inducible beta-lactamases (IBL) in clinical strains of gram-negative rods]. / Wystepowanie beta-laktamaz o rozszerzonym spektrum substratowym (ESBL) i beta-laktamaz indukowanych (IBL) u klinicznych szczepów paleczek Gram-ujemnych.

This study was undertaken to check the situation concerning the occurrence of Gram-negative rods producing extended-spectrum beta-lactamases (ESBL) and inducible beta-lactamases (IBL) in clinical specimens from patients hospitalized in National Clinical Hospital No. 1 in Warsaw. Such determinations were not performed in this hospital so far. During three months (April-June, 1997) 200 strains of Gram-negative rods were cultured. The strains were identified in automatic ATB system using strips with biochemical tests: ID 32 E for enteric rods and ID 32 GN for non-fermenting rods. ESBL-producing strains were detected with double disc diffusion test according to Jarlier et al. (1988). Clavulanate was applied as the inhibitor of beta-lactamases (AMO/CLAV disc). Inducible beta-lactamases were determined using double disc method according to Sanders and Sanders (1979). Cefoxitin was the inductor of these beta-lactamases. 82 strains (41% of all strains) belonging to Enterobacteriaceae family, 92 strains (46%) of Pseudomonadaceae rods and 26 strains (13%) of other Gram-negative rods were isolated. 30 ESBL-producing strains (15% of all strains) and 45 strains (22.5%) with IBL activity were detected. The obtained results confirm the necessity of continuous and reliable monitoring of ESBL--and IBL--producing strains among Gram-negative rods isolated from clinical materials. The aims of such procedure are the control and prevention of their dissemination within a hospital as well as the avoidance of therapeutic failures.

Prolyl endopeptidase from Sphingomonas capsulata: isolation and characterization of the enzyme and nucleotide sequence of the gene.

Prolyl endopeptidase (prolyl oligopeptidase, EC 3.4.21.26) was purified from Sphingomonas capsulata IFO 12533, and its gene was cloned and expressed in Escherichia coli. The recombinant enzyme was markedly inhibited by diisopropyl phosphofluoridate and hardly affected by SH reagents or metal chelators, similar to the native enzyme purified from S. capsulata. Nucleotide sequencing analysis revealed an open reading frame of 2169 bp, coding for a protein of 723 amino acids with a predicted molecular weight of 78,433. The amino acid sequence was 39.6, 45.3, 38.9, and 38.3% homologous to Flavobacterium meningosepticum, Aeromonas hydrophila, porcine brain, and human T cell prolyl endopeptidase, respectively. A region near the C-terminus and the region containing the putative catalytic triad residues were highly conserved. The enzyme was crystallized by the hanging drop vapor diffusion method, using ammonium sulfate as a precipitant.

Characterization of the genes encoding the three-component membrane-bound alcohol dehydrogenase from Gluconobacter suboxydans and their expression in Acetobacter pasteurianus.

The three-component membrane-bound alcohol dehydrogenase (ADH) of Gluconobacter suboxydans IFO12528 was purified, and the NH2-terminal amino acid sequence of each subunit was determined. On the basis of the amino acid sequences, the genes adhA, encoding the 72-kDa dehydrogenase, adhB, encoding the 44-kDa cytochrome c-553 (a CO-binding cytochrome c), and adhS, encoding a 15-kDa protein, were cloned and the amino acid sequences of their products were deduced from the nucleotide sequences. The dehydrogenase and cytochrome genes were clustered with the same transcription polarity, as is the case in species of Acetobacter, another genus of acetic acid bacteria. These AdhA and AdhB subunits showed similarity in amino acid sequence to those from Acetobacter spp., whereas AdhS showed no similarity to the corresponding subunit of the ADH complex of Acetobacter pasteurianus. Consistent with this, adhS of G. suboxydans could not complement a defect in the corresponding subunit of A. pasteurianus. When the adhA-adhB gene cluster of G. suboxydans was expressed in an ADH-deficient mutant of A. pasteurianus, the transformant showed distinct ADH activity. The ADH complex was purified to near homogeneity and consisted of two subunits, the dehydrogenase and the cytochrome c subunits derived from G. suboxydans, without any other subunit. These data suggested that AdhS, the smallest subunit of ADH, from G. suboxydans is not essential for ADH activity in A. pasteurianus, in contrast to the essential role of A. pasteurianus AdhS, which is required for correct assembly of the dehydrogenase and cytochrome c subunits on the membrane.

Measurements of oxidoreductase-like activity of intact bacterial cells by an amperometric method using a membrane-coated electrode.

The oxidation of D-glucose and nicotinic acid by intact cells of Gluconobacter industrious and Pseudomonas fluorescens, respectively, is successfully measured by an amperometric method using such compounds as Fe(CN)6(3-), p-benzoquinone, and dichlorophenolindophenol as electron acceptors. Analysis of the experimental results reveals that the intact cells behave like oxidoreductases whose kinetics follows a Michaelis-Mententype equation. The catalytic behavior is explained by a model which treats the bacterial cells as bags of enzymes and assumes distribution equilibrium in the concentrations of both the substrate and the electron acceptor between the test solution and the medium within the cells. The catalytic activity can be characterized by three quantities: the maximum reaction rate (vB) and the ratios of the Michaelis constant to the distribution constant for the substrate (Ks,cell/Ks,p) and to that for the electron acceptor (KM,cell/KM,p). Advanced modification of the model to involve the membrane permeability reveals that the three quantities are effective for explaining the catalytic behavior even when the permeability effect is significant. Thus, the three quantities should be regarded as the parameters which can reflect the permeability effect.

Use of a polymerase-chain-reaction-amplified DNA probe from Pseudomonas putida to detect D-hydantoinase-producing microorganisms by direct colony hybridization.

Pseudomonas putida strain DSM 84 produces N-carbamyl-D-amino acids from the corresponding D-5-monosubstituted hydantoins. The sequence of the D-hydantoinase gene from this strain (GenBank accession number L24157) was used to develop a DNA probe of 122 base pairs (bp) that could detect D-hydantoinase genes in other bacterial genera by DNA and by colony hybridization. Under conditions tolerating 32% mismatch, the probe was specific for all strains that expressed D-hydantoinase activity. These include Pseudomonadaceae of all rRNA groups, and bacteria belonging to the genera Agrobacterium, Serratia, Corynebacterium, and Arthrobacter. Environmental sampling was simulated by screening a mixture of unknown microorganisms from commercial inocula for the biodegradation of industrial, municipal and domestic wastes. The 122-bp probe was specific for microorganisms that subsequently demonstrated D-hydantoinase activity. Bacterial species from four different genera were detected, which were Pseudomonas, Klebsiella, Enterobacter, and Enterococcus.

[Features of secretion of bacteriolytic enzymes and polysaccharides in bacteria from the Pseudomonadaceae family]. / Osobennosti sekretsii bakterioliticheskikh fermentov i polisakharida u bakterii iz semeistva Pseudomonadaceae.

It is shown that secretion of bacteriolytic complex enzymes in a bacterium of the family Pseudomonadaceae begins in the latent and early logarithmic culture growth phases. A maximum of specific bacteriolytic activity falls in the same time interval with a maximum of specific activity of lytic proteinase L2 and muramidase. The activity of concomitant enzymes--non-specific proteinase and neutral phosphatase--peaks in the middle of the logarithmic growth phase. Secretion of exopolysaccharide into the external medium begins in the second half of the logarithmic growth phase and, thus, is not coordinated with secretion of bacteriolytic enzymes. It is shown that relative content of enzymes and polysaccharides depends upon the composition of growth medium for the producer.

Biosynthetic thiolase from Zoogloea ramigera. Mutagenesis of the putative active-site base Cys-378 to Ser-378 changes the partitioning of the acetyl S-enzyme intermediate.

The proposed active-site base Cys-378 of thiolase, responsible for deprotonation of acetyl-CoA, has been converted to a less acidic residue Ser-378 by mutagenesis. Comparison of the CD spectra and dimethyl suberimidate cross-linking experiments of the wild type, mutant Ser-378, and Gly-378 enzymes indicated that there have been no major conformational changes. The Ser-378 enzyme retains 0.1% of the Vmax of wild type in the direction of acetoacetyl-CoA thiolytic cleavage and 0.07% of the Vmax in the Claisen condensation direction. Analysis of the acetyl S-enzyme intermediate partitioning, that is capture of the acetyl enzyme by 1) the thiolate of coenzyme A relative to 2) the C-2 carbanion of acetyl-CoA, is changed to favor reaction 2 in the case of the Ser-378 mutant enzyme.

NADPH regeneration by glucose dehydrogenase from Gluconobacter scleroides for l-leucovorin synthesis.

A new process for (6S)-tetrahydrofolate production from dihydrofolate was designed that used dihydrofolate reductase and an NADPH regeneration system. Glucose dehydrogenase from Gluconobacter scleroides KY3613 was used for recycling of the cofactor. The reaction mixture contained 200 mM dihydrofolate, 220 mM glucose, 2 mM NADP, 14.4 U/ml dihydrofolate reductase, and 14.4 U/ml Glucose dehydrogenase, and the reaction was complete after incubation at pH 8.0, and 40 degrees C for 2.5 hr. With (6S)-tetrahydrofolate as the starting material, l-leucovorin was synthesized via a methenyl derivative. The purity of the l-leucovorin was 100%, and its diastereomeric purity was greater than 99.5% d.e. as the (6S)-form.