Current opinion on the prospect of mapping electronic orbitals in the transmission electron microscope: State of the art, challenges and perspectives.

The concept of electronic orbitals has enabled the understanding of a wide range of physical and chemical properties of solids through the definition of, for example, chemical bonding between atoms. In the transmission electron microscope, which is one of the most used and powerful analytical tools for high-spatial-resolution analysis of solids, the accessible quantity is the local distribution of electronic states. However, the interpretation of electronic state maps at atomic resolution in terms of electronic orbitals is far from obvious, not always possible, and often remains a major hurdle preventing a better understanding of the properties of the system of interest. In this review, the current state of the art of the experimental aspects for electronic state mapping and its interpretation as electronic orbitals is presented, considering approaches that rely on elastic and inelastic scattering, in real and reciprocal spaces. This work goes beyond resolving spectral variations between adjacent atomic columns, as it aims at providing deeper information about, for example, the spatial or momentum distributions of the states involved. The advantages and disadvantages of existing experimental approaches are discussed, while the challenges to overcome and future perspectives are explored in an effort to establish the current state of knowledge in this field. The aims of this review are also to foster the interest of the scientific community and to trigger a global effort to further enhance the current analytical capabilities of transmission electron microscopy for chemical bonding and electronic structure analysis.

Imaging the Spatial Distribution of Electronic States in Graphene Using Electron Energy-Loss Spectroscopy: Prospect of Orbital Mapping.

The spatial distributions of antibonding π^{*} and σ^{*} states in epitaxial graphene multilayers are mapped using electron energy-loss spectroscopy in a scanning transmission electron microscope. Inelastic channeling simulations validate the interpretation of the spatially resolved signals in terms of electronic orbitals, and demonstrate the crucial effect of the material thickness on the experimental capability to resolve the distribution of unoccupied states. This work illustrates the current potential of core-level electron energy-loss spectroscopy towards the direct visualization of electronic orbitals in a wide range of materials, of huge interest to better understand chemical bonding among many other properties at interfaces and defects in solids.

Finite-element-modeling of egg white as a substitute for tissue coagulation during bipolar radiofrequency-induced thermofusion.

Radiofrequency-induced thermofusion is a frequently used electrosurgical procedure for the sealing of blood vessels. A disadvantage of vessel sealing instruments is that the generated thermal energy spreads to the surrounding tissue and may irreversibly damage it. This is particularly problematic when operating close to sensitive structures such as nerves. Given their advantages, there is nonetheless a lot of interest in using bipolar vessel sealing for surgical procedures. To select instruments that may be safely used in such cases, it is important to reliably quantify the thermal spread to the surrounding tissue. Mathematical models can help to evaluate the transient behavior, that is the evolution of the thermal spread over time, more precisely. A finite element model allows for a detailed analysis of inhomogeneities in the spatial temperature distribution. As a first step towards a finite model of the bipolar vessel sealing process, a model of the coagulation of chicken egg white is presented here. Egg white has thermal and electrical properties that are very similar to tissue, making it suitable as a substitute for the analysis of the coagulation process. It has the additional advantage, that the spatial and temporal evolution of the thermal spread can be visually gauged. The presented model describes the experimentally observed spatial temperature distribution, the shape of the coagulated egg white, and the formation of hotspots. Furthermore, it is shown that the model can correctly predict the shape of the coagulated egg white in further experiments.

A mathematical model of bipolar radiofrequency-induced thermofusion.

Bipolar radiofrequency-induced thermofusion has become a widely accepted method successfully used in open and particularly in minimally-invasive surgery for the sealing of blood vessels and tissue of up to several millimeters diameter. Despite its wide-spread application, the thermofusion process itself is not well understood on a quantitative and dynamic level, and manufacturers largely rely on trial-and-error methods to improve existing instruments. To predict the effect of alternative generator control strategies and to allow for a more systematic approach to improve thermofusion instruments, a mathematical model of the thermofusion process is developed. The system equations describe the spatial and temporal evolution of the tissue temperature due to Joule heating and heat transfer, and the loss of tissue water due to vaporization. The resulting effects on the tissue properties, most importantly the electrical resistivity, heat capacity and thermal conductivity, are considered as well. Experimental results indicate that the extent of the lateral thermal damage is directly affected by Joule heating of the lateral tissue. The experimental findings are supported by simulation results using the proposed mathematical model of thermofusion.

Metagenomics-based analysis of viral communities in dairy lagoon wastewater.

Microbial populations, especially those of viruses, are poorly studied in dairy wastewater treatment operations. Here we report signature nucleic acid metagenomic sequences obtained by pyrosequencing viromes of virus-like particles that were extracted from two dairy waste treatment lagoons. The lagoons are operated in series, with Lagoon I being used as the primary stage and Lagoon II as the secondary stage of wastewater treatment. An average of 2000 sequences was obtained from each lagoon. More than 300 signatures from each lagoon matched sequences in the virus database of the National Center for Biotechnology Information (NCBI). We utilized a bioinformatics approach and transmission electron microscopy (TEM) to characterize the viral diversity and presence of potential viral pathogens within the lagoons. Our results showed differences in viral community compositions between Lagoon I and Lagoon II, suggesting that the viral community changes significantly in the transition of water between the two lagoons. Furthermore, the diverse viral community in the lagoon samples contained signature sequences of a variety of bacterial, plant, and animal viruses. Bacteriophage sequences dominated the viral community metagenomes in both lagoons. Ultimately these results can be used to identify viral bioindicators to rapidly assess wastewater treatment quality and the potential impacts of dairy operations on watersheds. Our viral metagenomic sequences have been submitted to GenBank (GPID 65805) and can provide insight into the composition and structure of viral communities within wastewaters of dairy lagoon systems.

Phylogeny of Sphingomonas species that degrade pentachlorophenol.

Four pentachlorophenol (PCP)-degrading bacteria isolated from geographically diverse areas have been examined in detail as regards their physiology and phylogeny. According to traditional biochemical methods, these strains had been classified as members of the genera Arthrobacter, Flavobacterium, Pseudomonas, and Sphingomonas. The PCP degradation pathway has been studied extensively in Sphingomonas (Flavobacterium) sp strain ATCC 39723 and the first three degradation steps catalyzed by a PCP-4-monooxygenase (PcpB) and a reductive dehalogenase (PcpC) that functions twice are well established. A fourth step appears to involve ring-fission of the aromatic nucleus (PcpA). Molecular analyses revealed that the PCP degradation pathway in these four strains was rather conserved, leading to a phylogenetic analysis using 16S rDNA. The results revealed a much closer phylogenetic relationship between these organisms than traditional classification indicated, placing them into the more recently established genus Sphingomonas where they may even represent a single species. With 16S rDNA analysis, many bacterial isolates involved in degradation of xenobiotic compounds that were previously classified into diverse genera have been reclassified into the genus Sphingomonas.

2,4,6-Trinitrotoluene (TNT) transformation by clostridia isolated from a munition-fed bioreactor: comparison with non-adapted bacteria.

Several bacterial strains were examined for their ability to degrade the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT). The strains examined included various clostridial strains isolated from a 4-year-old munition enrichment, related clostridial strains obtained from a culture collection, two enteric bacteria, and three lactobacilli. All Clostridium species tested were able to reduce TNT rapidly in a complex medium. In cell suspension experiments, these strains were also able to reduce 2,4-diamino-6-nitrotoluene (DANT) to 2,4,6-triaminotoluene (TAT) and to produce a compound that is not yet identified; thus, they could not be distinguished from one another with regard to the pathway of transformation. The enteric strains and the lactobacilli were able to perform the initial reduction of TNT, but none was capable of reducing DANT in cell suspensions.

PCP degradation is mediated by closely related strains of the genus Sphingomonas.

There have been numerous reports in the literature of diverse bacteria capable of degrading pentachlorophenol (PCP). In order to gain further insight into the phylogenetic relationships of PCP-degrading bacteria, we examined four strains: Arthrobacter sp. strain ATCC 33790, Flavobacterium sp. strain ATCC 39723, Pseudomonas sp. strain SR3, and Sphingomonas sp. strain RA2. These organisms were isolated from different geographical locations and all of them degrade high concentrations (100-200 mg/L) of PCP. Southern blot analyses determined that these bacteria all harbour DNA that encodes similar, if not identical, genes involved in PCP degradation. Comparison of the 16S rRNA nucleotide sequences revealed that these organisms were very closely related and, in fact, represent a monophyletic group. The 16S rRNA analyses together with fatty acid and sphingolipid analyses strongly suggest that the four strains are members of the genus Sphingomonas. The close relationship of the four organisms is supported by nucleotide sequence analysis data of the pcpB locus encoding PCP-4-monooxygenase, the first enzyme in the PCP degradative pathway.

Nucleotide sequence of the transcriptional control region of the osmotically regulated proU operon of Salmonella typhimurium and identification of the 5' endpoint of the proU mRNA.

Southern blot analysis of 15 proU transposon insertions in Salmonella typhimurium indicated that this operon is at least 3 kilobase pairs in length. The nucleotide sequence of 1.5-kilobase-pair fragment that contains the transcriptional control region of the proU operon and the coding sequences specifying 290 amino acids of the first structural gene of the operon was determined. The predicted amino acid sequence of the product of this gene shows extensive similarity to the HisP, MalK, and other proteins that are inner membrane-associated components of binding protein-dependent transport systems. S1 mapping and primer extension analysis of the proU mRNAs revealed several species with different 5' ends. Two of these endpoints are sufficiently close to sequences that have weak similarities to the consensus -35 and -10 promoter sequences that they are likely to define two transcription start sites. However, we cannot rule out the possibility that some or all of the 5' endpoints detected arose as a result of the degradation of a longer mRNA. The expression of proU-lacZ operon fusions located on plasmids was normal in S. typhimurium regardless of the plasmid copy number. The sequences mediating normal, osmoregulated expression of the proU operon were shown by subcloning to be contained on an 815-base-pair fragment. A 350-base-pair subclone of this fragment placed onto a lacZ expression vector directed a high-level constitutive expression of beta-galactosidase, suggesting that there is a site for negative regulation in the proU transcriptional control region which has been deleted in the construction of this plasmid.

Evaluation of Hemophilus type B systemic isolates for beta-lactamase and non-beta-lactamase mediated ampicillin resistance and for susceptibility to other antimicrobial agents.

Of 175 recent Minnesota Hemophilus influenzae type b isolates from systemic disease, 43 were found to be resistant to ampicillin (greater than or equal to 4 micrograms/mL [mg/L]), each of which produced beta-lactamase. Of the 132 ampicillin-susceptible isolates, 68 (52%), all beta-lactamase negative, had minimum inhibitory concentrations (MICs) of either 1 or 2 micrograms/mL (mg/L), indicating relative resistance if derived from cerebrospinal fluid (CSF) infections. From a review of the literature, and in agreement with the authors findings, ampicillin-resistant beta-lactamase-negative isolates are rare and are likely to be nontypeable, of respiratory origin, and with MICs in the low resistance range. For the 43 ampicillin-resistant isolates, percentages resistant to other agents were as follows: 0% chloramphenicol, 0% rifampin, 6% tetracycline, 0% trimeprim-sulfamethoxazole, 2% cefamandole, 5% cefaclor, 2% moxalactam, and 0% for the remaining third-generation cephalosporins cefotaxime, ceftazidime, and ceftizoxime. Unlike ampicillin-resistant isolates, 100% of ampicillin-susceptible isolates had relatively low cefaclor MICs of less than or equal to 4 micrograms/mL (mg/L), suggesting a relatively increased H. influenzae beta-lactamase effect on cefaclor in comparison with the other cephalosporins tested.

Oxacillin killing curve patterns of Staphylococcus aureus isolates by agar dilution plate count method.

The bactericidal dynamics of oxacillin against four Staphylococcus aureus isolates with known 24-h "persister" percentages were studied by using the agar dilution plate count method. Isolates were selected to provide a representative spectrum whose individual 24-h trough intrinsic persister percentages ranged from greater than 1 to less than 0.01%. Resultant agar dilution plate count method killing curve patterns were found to be reproducible and served to characterize each isolate. The paradoxical effect was observed for each isolate, with paradoxical peaks tending to develop and diminish sequentially during the course of oxacillin action. The observed strain-dependent dynamics of oxacillin killing underscore the artifactual nature of the so-called tolerance phenomenon and negate the usefulness of MBCs and MBC/MIC ratios for the characterization of S. aureus isolates.

Influence of technical factor variations during inoculum preparation on the agar dilution plate-count method for quantitation of Staphylococcus aureus oxacillin persisters.

The possible introduction of error into Staphylococcus aureus oxacillin intrinsic persister measurements by the agar dilution plate-count method resulting from technical factor variations associated with inoculum preparation was investigated. pH stability and exponential growth were shown to be present at the time of inoculum standardization. The use of glass or plastic tubes and the contamination of tube walls during inoculum broth culture produced no differences in test results. For S. aureus and oxacillin, the agar dilution plate-count method is a new and reliable approach to the quantitative study of bacterial inhibition and killing.

Evaluation of the AutoMicrobic system Gram-Positive Susceptibility-MIC card for detection of oxacillin-resistant coagulase-negative staphylococci.

A total of 398 consecutive clinical staphylococcus isolates, of which 205 were coagulase negative and 193 were coagulase positive, were tested in parallel by using AutoMicrobic system Gram-Positive Susceptibility-MIC cards and modified Mueller-Hinton agar containing 4% NaCl and oxacillin (6 micrograms/ml). The AutoMicrobic system cards correctly detected 103 of 104 (99%) oxacillin-resistant coagulase-negative isolates with no reports of false resistance.

Evaluation of the newly modified AutoMicrobic system gram-positive susceptibility-MIC card for detection of methicillin-resistant Staphylococcus aureus.

AutoMicrobic system (Vitek Systems, Inc., Hazelwood, Mo.) gram-positive susceptibility-MIC (GPS-MIC) cards and GPS cards were compared for their ability to detect methicillin-resistant Staphylococcus aureus (MRSA) in two groups of isolates. MRSA isolates from the first group were detected at rates of 23 and 85%, and MRSA isolates from the second group were detected at rates of 95 and 88% for GPS and GPS-MIC cards, respectively. Detection of MRSA by the AutoMicrobic system lacks sensitivity and remains unsatisfactory unless accompanied by supplemental testing.

Evaluation of mannitol salt agar with oxacillin as a screening medium for methicillin-resistant Staphylococcus aureus.

We evaluated the use of mannitol salt agar with oxacillin for use as a primary screening medium for the simultaneous detection and identification of methicillin-resistant Staphylococcus aureus in clinical surveillance specimens. Oxacillin agar dilution susceptibility tests with mannitol salt agar and Mueller-Hinton agar were performed in parallel with disk-agar diffusion testing on 95 oxacillin-susceptible and 105 oxacillin-resistant S. aureus stock isolates. MICs were found to be comparable, showing distinct separation of susceptible and resistant isolates into two groups with MICs of less than or equal to 2 and greater than or equal to 32 micrograms/ml, respectively. In accord with these findings, 4 micrograms of oxacillin per ml was selected for use in the screening medium. For performance evaluation, mannitol salt agar with 4 micrograms of oxacillin per ml was compared with mannitol salt agar without oxacillin by performing parallel screening tests on 153 clinical surveillance specimens. For detection of methicillin-resistant S. aureus, mannitol salt agar with 4 micrograms of oxacillin per ml was as sensitive as mannitol salt agar without oxacillin and required significantly fewer confirmatory tests. For primary identification of methicillin-resistant S. aureus, mannitol salt agar with 4 micrograms of oxacillin per ml was 6.4% false-positive and 1.1% false-negative, with a 93.6% positive predictive value. These findings indicate that mannitol salt agar with 4 micrograms of oxacillin per ml can be used as a reliable and cost-effective screening medium for the simultaneous detection and identification of methicillin-resistant S. aureus in clinical surveillance specimens.

Evaluation of oxacillin tolerance in Staphylococcus aureus by a novel method.

A novel agar dilution plate-count procedure for the quantitative measurement of bacterial inhibition and killing is described. For Staphylococcus aureus versus oxacillin, by the agar dilution plate-count procedure it was found that only 1 of 20 clinical isolates and 1 of 7 allegedly tolerant reference isolates met the conventional definition of tolerance. By using inocula of 10(5) CFU per plate, most isolates were demonstrated to have subpopulations of cells which, although inhibited, persisted for 24 h in concentrations significantly above their MICs. The persister percentages at 24 h appeared to be strain dependent, and all persisters exhibited the paradoxical effect. For each isolate, the persister percentage markedly decreased after action by oxacillin for 48 h, and the paradoxical effect was greatly diminished. Our findings suggest that tolerance is an artificial and arbitrary concept that does not adequately characterize the inhibition and killing dynamics associated with the persister phenomenon.

Evaluation of the AutoMicrobic system for identification and susceptibility testing of gram-negative bacilli.

The AutoMicrobic system (AMS) (Vitek Systems, Inc., Hazelwood, Mo.) was compared with the API-20E system for the identification of gram-negative bacilli by using 380 stock clinical isolates and 377 immediately encountered fresh clinical isolates. For the stock isolates, with Enterobacteriaceae-Plus Biochemical Cards and automated interpretation, 364 (95.8%) were in agreement to the species level. For the fresh clinical isolates, agreement at the genus and species levels was 89.7 and 85.9%, respectively, when Enterobacteriaceae-Plus Cards were interpreted by the AMS. Manual interpretation of Enterobacteriaceae-Plus Biochemical Cards improved species level agreement to 91.0%. Subsequent retesting of all discrepant isolates with the Gram-Negative Identification Card resulted in significant improvement of results, and for the stock and fresh clinical isolates, species level agreement was 98.7 and 97.3%, respectively. AMS susceptibility testing was evaluated by comparing ampicillin and cephalothin MICs determined in parallel by AMS and a reference broth microdilution test for stock isolates, and by comparison of AMS and standardized disk agar diffusion test results for fresh clinical isolates. For the stock isolates, AMS mean integer MICs approximated microdilution mean integer MICs with AMS, providing excellent MIC replicability. For ampicillin and cephalothin, 50 and 46.8%, respectively, of AMS integer MICs were within +/- 1 microgram/ml of the reference values, and 89.3 and 63.1% of AMS integer MICs were within +/- 2 micrograms/ml of the reference values. For the fresh clinical isolates, AMS and reference results were in disagreement for 4.5% of the antimicrobial agents tested, with 2.3% as a combination of "major" and "very major" errors.

Antimicrobial resistance in Haemophilus isolates: a Minnesota experience and literature review.

Annual ampicillin susceptibility rates for Haemophilus influenzae isolates at the St. Paul-Ramsey Medical Center gradually decreased from 100% in 1974 to 83.3% in 1980 and then remained stable at 88.90%. Penicillin susceptibility rates were similar to those for ampicillin. Ampicillin rates were source dependent: eye 95%, respiratory 90%, miscellaneous sources 82%, and blood and CSF 80%. Rates for Haemophilus parainfluenzae varied and showed no trend. H. parainfluenzae isolates were distinctly less susceptible to penicillin (70%) than to ampicillin (96%). H. influenzae isolates were highly susceptible to chloramphenicol (99.6%) and tetracycline (97.5%), with the latter also showing source dependency. Characterization of isolates for colony morphology and hemolysis showed no clinical relevancy. Ampicillin and penicillin MICs were determined for 128 clinical isolates saved in stock culture during 1978-1983. All 19 resistant isolates (MIC greater than or equal to 4 micrograms/mL) were resistant to both penicillin and ampicillin and produced beta-lactamase. Eight had penicillin MICs of 1 or 2 micrograms/mL and three had ampicillin MICs of 1 or 2 micrograms/mL. The significance of isolates with MICs of 1-2 micrograms/mL is discussed in relation to our findings and a review of the literature.

Evaluation of the automicrobic system for detection of resistance of Staphylococcus aureus to methicillin.

The AutoMicrobic system (AMS) (Vitek System, Inc., Hazelwood, Mo.) was tested for its ability to determine oxacillin and gentamicin susceptibility of 98 known oxacillin-susceptible and 103 known oxacillin-resistant Staphylococcus aureus isolates. AMS and reference oxacillin susceptibility results were in agreement for all 95 (100%) oxacillin-susceptible isolates. In contrast, only 23 (22.3%) of the 103 known oxacillin-resistant isolates were correctly reported. For the known oxacillin-resistant isolates, 65 received AMS reports at 3 to 4 h, with only 9% being correct, whereas 38 were reported at 5 to 6 h, with 47% being correct. The reliability of AMS gentamicin susceptibility results was evaluated by testing the 198 S. aureus isolates in parallel with MIC-2000 broth dilution tests. AMS gentamicin susceptibility results were found to be reliable and essentially identical to MIC-2000 results. The possibility of improving AMS oxacillin resistance detection by using gentamicin resistance as a linked screening marker for oxacillin resistance was evaluated with data from the parallel AMS and MIC-2000 gentamicin susceptibility tests and from data accrued on recent clinical laboratory isolates. By these two approaches, respective sensitivities of 97 and 99.8%, and specificity of 72%, were found for detection of oxacillin-resistant isolates by using gentamicin resistance as a marker.

Evaluation of the automicrobic system for susceptibility testing of Pseudomonas aeruginosa to gentamicin, tobramycin, and amikacin.

The AutoMicrobic system (AMS; Vitek Systems, Inc., Hazelwood, Mo.) was studied for its ability to produce accurate and precise MIC interpretations for Pseudomonas aeruginosa susceptibility to gentamicin, tobramycin, and amikacin. MICs were determined in parallel on 200 selected P. aeruginosa isolates by using the AMS discrete-integer MIC program AMS p12.ROB for interpretation of the AMS Gram-Negative General Susceptibility Urinary Card, and a reference small-integer broth microdilution test. Parallel AMS and broth microdilution MICs were also replicated for three selected strains of P. aeruginosa for which MICs were representative of the dilution test ranges. For the 200 P. aeruginosa isolates, mean AMS MICs were significantly larger than the reference test mean MICs, coefficients of variation were approximately double those of the reference test, and correlation coefficients were unacceptably low for each antimicrobial agent. MIC replication studies for the three selected P. aeruginosa strains showed comparable AMS and reference mean MICs in the lower portions of the dilution ranges, significantly higher AMS mean MICs in the upper portions, and mean coefficients of variation of 63 and 9.6%, respectively, for replicated AMS and reference MICs. These results indicate that the AMS, in its present stage of development, does not produce acceptable MIC measurements for P. aeruginosa susceptibility to gentamicin, tobramycin, and amikacin.