Comparative evaluation of As, Se and V removal technologies for the treatment of oil refinery wastewater.

In this study, a broad range of readily deployable metal removal technologies were tested on a US refinery's wastewater to determine vanadium, arsenic and selenium removal performance. The bench-scale treatability studies were designed and performed so that test conditions could be as uniform as possible given the different mechanisms of action and engineering applications of each technology. The experimental data show that both ferric precipitation and reactive filtration were able to remove As, Se and V more efficiently from the wastewater than other tested technologies. Additionally, granular ferric hydroxide (GFH) adsorption was also effective in both V and As removal. Although the thiol-SAMMS adsorbent was developed for mercury removal, it also demonstrated appreciable selenium removal. None of the tested membrane filtration technologies showed any significant metals removal. This was attributed to the dissolved form of the metals as well as the wastewater's fouling characteristics.

Structure-based design and in-parallel synthesis of inhibitors of AmpC beta-lactamase.

BACKGROUND: Group I beta-lactamases are a major cause of antibiotic resistance to beta-lactams such as penicillins and cephalosporins. These enzymes are only modestly affected by classic beta-lactam-based inhibitors, such as clavulanic acid. Conversely, small arylboronic acids inhibit these enzymes at sub-micromolar concentrations. Structural studies suggest these inhibitors bind to a well-defined cleft in the group I beta-lactamase AmpC; this cleft binds the ubiquitous R1 side chain of beta-lactams. Intriguingly, much of this cleft is left unoccupied by the small arylboronic acids. RESULTS: To investigate if larger boronic acids might take advantage of this cleft, structure-guided in-parallel synthesis was used to explore new inhibitors of AmpC. Twenty-eight derivatives of the lead compound, 3-aminophenylboronic acid, led to an inhibitor with 80-fold better binding (2; K(i) 83 nM). Molecular docking suggested orientations for this compound in the R1 cleft. Based on the docking results, 12 derivatives of 2 were synthesized, leading to inhibitors with K(i) values of 60 nM and with improved solubility. Several of these inhibitors reversed the resistance of nosocomial Gram-positive bacteria, though they showed little activity against Gram-negative bacteria. The X-ray crystal structure of compound 2 in complex with AmpC was subsequently determined to 2.1 A resolution. The placement of the proximal two-thirds of the inhibitor in the experimental structure corresponds with the docked structure, but a bond rotation leads to a distinctly different placement of the distal part of the inhibitor. In the experimental structure, the inhibitor interacts with conserved residues in the R1 cleft whose role in recognition has not been previously explored. CONCLUSIONS: Combining structure-based design with in-parallel synthesis allowed for the rapid exploration of inhibitor functionality in the R1 cleft of AmpC. The resulting inhibitors differ considerably from beta-lactams but nevertheless inhibit the enzyme well. The crystal structure of 2 (K(i) 83 nM) in complex with AmpC may guide exploration of a highly conserved, largely unexplored cleft, providing a template for further design against AmpC beta-lactamase.

An extended-spectrum AmpC-type beta-lactamase obtained by in vitro antibiotic selection.

A predictive approach was assayed to evaluate the possibility of mutant Amp-C beta-lactamase emergence with increased substrate spectrum (including new C-3' quaternary ammonium cephems). The ampC gene encoding the AmpC beta-lactamase from Enterobacter cloacae was cloned and expressed in an AmpC-defective strain of E. coli. After the AmpC containing strain was challenged with cefpirome, an ampC variant encoding an enzyme with increased resistance to cefpirome and cefepime was selected. In addition, this variant conferred increased resistance to penicillins and third generation cephalosporins. The complete nucleotide sequence of the gene was determined. The deduced peptide sequence showed a single change with respect to the wild-type gene: valine to glutamic acid at position 318 of the native protein (298 of the mature enzyme). The potential emergence and spread of this type of AmpC variants among pathogens should be considered.

Strategies to minimize the development of antibiotic resistance.

The choice of appropriate antimicrobial agents should take into consideration not only the interests of the individual patient, but also the ecological impact of different drugs and their delivery schedules. Selection of antibiotic-resistant organisms is a key aspect to remember. Bacterial populations harboring determinants of antibiotic resistance will be selected for by a range of antibiotic concentrations which are able to suppress or slow the growth of susceptible populations. These concentrations (selective concentrations) will be achieved within the human body in a series of compartments (selective compartments), where the potential selective power will be roughly proportional to the time of exposure of the bacteria to the drug (selective period). The duration of the expected exposure of bacterial populations to these concentrations of the drugs and the number of challenges they experience are probably the most important factors in predicting the potential selective activity of an antibiotic regimen. Such a risk analysis procedure may be used to propose guidelines for minimizing the development of antibiotic resistance.

[National multicenter study of the in vitro activity of moxifloxacin against respiratory tract pathogens. Spanish Study Group on Moxifloxacin]. / Estudio multicéntrico nacional de la actividad in vitro de moxifloxacino frente a patógenos respiratorios. Grupo Español de Estudio de Moxifloxacino]

The activity of moxifloxacin, a novel 8-methoxyquinolone, was evaluated against 1,218 respiratory pathogens isolated in nine Spanish hospitals and was compared with ciprofloxacin, sparfloxacin, amoxicillin, amoxicillin-clavulanic acid, cefuroxime, erythromycin and clarithromycin. Moxifloxacin exhibited an excellent in vitro activity against most tested isolates with MIC90 values of 0.25 mg/l for Streptococcus pneumoniae and viridans group streptococci; 0.12 mg/l for Streptococcus pyogenes; 0.25 mg/l for Streptococcus agalactiae; 0.06 and 4 mg/l for methicillin-susceptible and -resistant Staphylococcus aureus, respectively; 0.06 mg/l for Haemophilus influenzae and 0.12 mg/l for Moraxella catarrhalis. Moxifloxacin susceptibility rates were not affected by penicillin resistance in S. pneumoniae and S. viridans, by the betalactamase production in H. influenzae and M. catarrhalis or by macrolide resistance. Moxifloxacin was twice as active as sparfloxacin and four to sixteen times more active than ciprofloxacin against Gram-positive isolates. Sparfloxacin and ciprofloxacin were slightly more active than moxifloxacin when tested against H. influenzae and M. catarrhalis isolates. The microbiological data obtained confirm that moxifloxacin is a promising antimicrobial agent for treating respiratory tract infections.

Selective removal of plutonium 238 from a canal sediment using a carbonate-chelant soil washing technology (ACT*DE*CON).

The Mound laboratory site in Miamisburg, OH, a former plutonium processing facility, contains approximately 40000 yd(3) (30,580 m3) of plutonium- and thorium-contaminated soils and sediments at levels that require remediation. Existing applicable remediation technologies are unsatisfactory, because they are expensive and do not provide volume reduction. ACT*DE*CON is a chemical soil leaching technology for the treatment of soils that utilizes contaminant dissolution via dilute selective solutions to remove radionuclides. In bench-scale tests, process parameters were developed for the optimal treatment of the Miami Erie Canal soil at the Mound site, combining the maximum plutonium removal with an acceptable amount of soil dissolution and minimizing the costs of reagents. Parameters evaluated included soil to extractant mass ratio, temperature, rinse solution composition, kinetics, and the application of several dewatering aids. Plutonium removal rates of >95% were achieved, and the residual plutonium in the treated soil proved to be very immobile-confirming that the process had removed the most accessible species of the radionuclide. Currently being tested at Mound is an engineering scale-up that includes an attrition scrubber, a counter-current extractor, and a reverse osmosis system. Economic evaluations based on bench-scale results put the treatment cost at US$278/yd(3) (US$364/m3), compared to US$350/yd(3) (US$458/m3) for the 'box-and-bury' baseline alternative treatment system.

An attempt towards simultaneous biobased solvent based extraction of proteins and enzymatic saccharification of cellulosic materials from distiller's grains and solubles.

Distiller's grains and solubles (DGS) is the major co-product of corn dry mill ethanol production, and is composed of 30% protein and 30-40% polysaccharides. We report a strategy for simultaneous extraction of protein with food-grade biobased solvents (ethyl lactate, d-limonene, and distilled methyl esters) and enzymatic saccharification of glucan in DGS. This approach would produce a high-value animal feed while simultaneously producing additional sugars for ethanol production. Preliminary experiments on protein extraction resulted in recovery of 15-45% of the protein, with hydrophobic biobased solvents obtaining the best results. The integrated hydrolysis and extraction experiments showed that biobased solvent addition did not inhibit hydrolysis of the cellulose. However, only 25-33% of the total protein was extracted from DGS, and the extracted protein largely resided in the aqueous phase, not the solvent phase. We hypothesize that the hydrophobic solvent could not access the proteins surrounded by the aqueous phase inside the fibrous structure of DGS due to poor mass transfer. Further process improvements are needed to overcome this obstacle.

Selective compartments for resistant microorganisms in antibiotic gradients.

The development of bacterial resistance to antibiotics is one of the best documented examples of contemporary biological evolution. Variability in the mechanisms of resistance depends on the diversity of genotypes in the huge bacterial populations, and also on the diversity of selective pressures that are produced along the antibiotic concentration gradients formed in the highly compartmentalized human body during therapy. These antibiotic gradients can be conceived as comprising selective compartments, each one of them defined as the concentration able to select a particular genetic variant. In vitro experimental models confirm that some antibiotic resistant variants are selected only at certain selective concentrations of antibiotics. The correspondence between selective compartments and selectable variants could offer a way of describing more accurately the antibiotic selective landscapes and for taking measures to prevent the development of a major threat to the future of modern medicine.

Concentration-dependent selection of small phenotypic differences in TEM beta-lactamase-mediated antibiotic resistance.

In this paper, the first robust experimental evidence of in vitro and in vivo concentration-dependent selection of low-level antibiotic-resistant genetic variants is described. The work is based on the study of an asymmetric competition assay with pairs of isogenic Escherichia coli strains, differing only (apart from a neutral chromosomal marker) in a single amino acid replacement in a plasmid-mediated TEM-1 beta-lactamase enzyme, which results in the new TEM-12 beta-lactamase. The mixture was challenged by different antibiotic concentrations, both in vitro and in the animal model, and the selective process of the variant population was carefully monitored. A mathematical model was constructed to test the hypothesis that measured growth and killing rates of the individual TEM variants at different antibiotic concentrations could be used to predict quantitatively the strength of selection for TEM-12 observed in competition experiments at these different concentrations.

Selection of naturally occurring extended-spectrum TEM beta-lactamase variants by fluctuating beta-lactam pressure.

Despite the large number of in vitro mutations that increase resistance to extended-spectrum cephalosporins in TEM-type beta-lactamases, only a small number occur in naturally occurring enzymes. In nature, and particularly in the hospital, bacteria that contain beta-lactamases encounter simultaneous or consecutive selective pressure with different beta-lactam molecules. All variants obtained by submitting an Escherichia coli strain that contains a bla(TEM-1) gene to fluctuating challenge with both ceftazidime and amoxicillin contained only mutations previously detected in naturally occurring beta-lactamases. Nevertheless, some variants obtained by ceftazidime challenge alone contained mutations never detected in naturally occurring TEM beta-lactamases, suggesting that extended-spectrum TEM variants in hospital isolates result from fluctuating selective pressure with several beta-lactams rather than selection with a single antibiotic.

Single amino acid replacements at positions altered in naturally occurring extended-spectrum TEM beta-lactamases.

By directed mutagenesis, we constructed a set of seven TEM-1 derivatives containing single replacements in each one of the amino acids substituted in naturally occurring extended-spectrum TEM beta-lactamases. The exact contribution of each mutation to the resistance phenotype was determined. In addition, mutant enzyme production and stabilities were studied. Five of seven mutations determined to some extent variations in cephalosporin and/or monobactam activity. Dramatic changes in the hydrolysis of ceftazidime and aztreonam occurred when a serine was at position 164. Changes at positions 104, 238, and 240 showed more leaky variation in activity towards cephalosporins and aztreonam. Replacements at positions 237 and 265 caused no variation in susceptibility to cephalosporins. Interestingly, the change from Gln to Lys at position 39 found in TEM-2, classically considered a neutral change, slightly but consistently increased the MIC of ceftazidime and aztreonam. The in vitro construction of mutations appearing in naturally occurring TEM-beta-lactamases, studied in the same genetic context, may help to understand the evolution of extended-spectrum beta-lactamases.

The antibiotic selective process: concentration-specific amplification of low-level resistant populations.

The biochemistry and genetics of antibiotic resistance are far better known than the equally important events underlying the selection of resistant populations. The hidden selection of low-level resistant variants may be a key process in the emergence of high-level antibiotic resistance. Different low-level resistant bacterial subpopulations may be specifically selected by different low antibiotic concentrations. The space in the environment (human body) where a given selective concentration exists represents the selective compartment. For pharmacokinetic reasons, low antibiotic concentrations occur in a larger selective compartment and persist longer than high antibiotic concentrations. The specific selection of low-level variants by low concentrations of antibiotic can be reproduced in experimental in vitro models using mixtures of susceptible and low-level resistant populations. We demonstrated this in Escherichia coli strains harbouring TEM-1, TEM-12 and TEM-10 beta-lactamases challenged by cefotaxime, and also Streptococcus pneumoniae strains with various levels of penicillin resistance challenged by amoxicillin or cefotaxime. In both cases, four hours of antibiotic challenge produced selective peaks of low-level resistant variant populations at low-level antibiotic concentrations. We conclude that variants with small decreases in antibiotic susceptibility may be fully selectable under in vivo circumstances; on the other hand, low-level antibiotic concentrations may have a considerable selective effect on the emergence of antibiotic resistance.

Characterization of a nosocomial outbreak involving an epidemic plasmid encoding for TEM-27 in Salmonella enterica subspecies enterica serotype Othmarschen.

A ceftazidime-resistant Salmonella enterica subspecies enterica serotype Othmarschen strain, harboring the plasmid-mediated new extended-spectrum beta-lactamase TEM-27, was involved in a nosocomial outbreak (8 patients) at the Pediatric Cardiology Department of the Ramón y Cajal Hospital in Madrid. Genomic DNA polymorphism analysis, using an arbitrarily primed polymerase chain reaction, demonstrated the clonal nature of all Salmonella isolates. The plasmid encoding TEM-27 (pJMM1) was characterized by EcoRI, SacI, and BglI restriction. An identical restriction pattern was found in plasmid from all S. enterica strains. Possible in vivo intergeneric plasmid spread was suggested by the identification of an identical plasmid in Escherichia coli RYC5H and Enterobacter cloacae RYC39737 strains isolated during the outbreak. Results indicate that this outbreak involved dissemination of a single ceftazidime-resistant Salmonella strain and the spread of a single TEM-27-encoding plasmid among different Enterobacteriaceae.

[Meropenem against bacteria carriers of wide spectrum TEM beta-lactamases: evolutive aspects]. / Meropenem frente a bacterias portadoras de betalactamasas TEM de amplio espectro: aspectos evolutivos.

The so-called wide spectrum beta-lactamases (WSBLs) are able to hydrolyze wide spectrum cephalosporins or monobactamics such as cefotaxime, ceftriaxone, ceftazidime, cefepime, cefpiroma or aztreonam. The natural wide spectrum beta-lactamases are mutational variants of TEM-1 consisting in the substitution of one of more amino acids within seven well defined positions in the molecule. Given the expected extremely low frequency for the simultaneous production of double or triple mutations, it is plausible that one of the mutational changes has been independently selected. A plurimutational remodelling of the TEM-beta-lactamase molecule is successively produced with the consequent appearance of highly effective ESBLs. Mutagenesis techniques allow clean molecular variants to be produced and allow the mutational effects under homogeneous conditions of bacterial strain, the plasmid implicated or the genic promotor to be studied. Meropenem remains active versus all the wide spectrum beta-lactamases referred in the 2be group of Bush, Jacoby and Medeiros as well as the new beta-lactamases produced in vitro by directed mutagenesis.

Selection of very small differences in bacterial evolution.

As the Science of Biology is constantly changing due to new discoveries and advanced techniques it is essential that a systematic study of the environmental causes of natural selection on microorganisms be conducted. Very small phenotypic differences among individuals within bacterial populations arise as a result of spontaneous genetic variation, but the evolutionary importance of these small changes is frequently considered to be non-significant. Recent in vitro experiments indicate that efficient selection of these very small differences may take place in environmental compartments where a particular intensity of the selective agent is exerted. Model studies based on competition between bacterial populations only differing in one or two amino acid changes of a detoxifying antibiotic enzyme (e.g. beta-lactamase) have shown that at a narrow range of antibiotic concentrations the variant population is strongly selected over the original type, despite the extremely low phenotypic differences in antibiotic susceptibility. These selective concentrations are expected to occur in precise environmental compartments (selective compartments). Due to the high frequency of structured habitats in natural environments, the intensity of selective agents is commonly exerted along certain gradients. Each one of the points forming these gradients (or intersection among gradients) may have a particular selective ability for a specific genetic variant. Considering the environment as a composition of an extremely high number of specific selective compartments may help to understand the existence of high levels of genetic variability in natural bacterial populations. This may be one of the clues towards the unraveling of bacterial evolution.

A237T as a modulating mutation in naturally occurring extended-spectrum TEM-type beta-lactamases.

A TEM-1 beta-lactamase derivative containing the single amino acid substitution A237T slightly increased (from 24 to 32 microg/ml) the cephalothin MIC for Escherichia coli RYC1000 but did not influence the activities of cefotaxime, ceftazidime, and aztreonam (MICs of 0.03, 0.12, and 0.06 microg/ml, respectively). Despite its apparent neutrality, addition of the A237T mutation to the pair of mutations characterizing TEM-10 (R164S and E240K) had a strong effect on substrate preference. Ceftazidime and aztreonam MICs decreased from 128 and 16 microg/ml to 16 and 2 microg/ml, respectively. In contrast, the cefotaxime MIC increased from 0.5 to 4 microg/ml. The acquisition of apparently neutral or even deleterious mutations results in a very effective mechanism of resistance to different beta-lactams that may be simultaneously or subsequently present in the environment. We propose here that the mutation in position 237 is an example of a modulating mutation and that consideration of this type of mutation may be important for understanding the evolution of beta-lactamases.

Antibiotic-selective environments.

The evolution and spread of antibiotic resistance depends on the antibiotic pressure exerted in the microbial environment. Selective effects occur in selective compartments, where particular antibiotic concentrations result in a differential growth rate of resistant bacterial variants. This may happen even at very low antibiotic concentrations able to select low-level-resistant bacteria. When more than one antibiotic is present in the environment, the multiple and fluctuating pressure produces the selection of bacterial variants that use multiple or multipurpose mechanisms or optimize a single mechanism of resistance to survive under the variable environmental conditions. Host factors such as immunity contribute to the selective process. Antibiotics themselves may promote bacterial diversity, either mediated by the random drift effect or triggering the increase of mutational events under bacterial stress. Analysis of selective environment-related antibiotic-host-bacteria interactions is essential to understanding the biology of antibiotic resistance.

In vitro selective antibiotic concentrations of beta-lactams for penicillin-resistant Streptococcus pneumoniae populations.

Therapeutic regimens containing beta-lactam antibiotics are selecting penicillin-resistant Streptococcus pneumoniae populations all over the world. The selective pressure after 4 h of exposure to different concentrations of amoxicillin, cefixime, cefuroxime, and cefotaxime for low-level or high-level penicillin-resistant S. pneumoniae was evaluated in an in vitro model with mixed populations with penicillin susceptibilities of 0.015, 0.5, 1, and 2 micrograms/ml. The antibiotic concentration selecting for low-level resistance strongly reduced the susceptible population. Increasing antibiotic concentrations tended to decrease the total proportion of penicillin-resistant bacteria because of reduced numbers of the low-level-resistant population. The antibiotic concentration selecting for high-level resistance produced fewer resistant populations, but most of the organisms selected represented high-level resistance. In general, amoxicillin was a good selector for the low-level-resistant population and a poor selector for high-level resistance; cefuroxime and cefotaxime were poor selectors for low-level resistance and better selectors than amoxicillin for high-level penicillin resistance. Cefixime was the best selector of low-level penicillin resistance. When only resistant populations were mixed, the strains with high-level resistance were selected even at low antibiotic concentrations. Determination of the effects of selective antibiotic concentrations on mixed cultures of bacteria expressing different antibiotic resistance levels may help researchers to understand the ecology and epidemiology of penicillin-resistant S. pneumoniae populations.