Raman spectroscopy for the microbiological characterization and identification of medically relevant bacteria

The detection and identification of pathogenic bacteria has become more important than ever due to the increase of potential bioterrorism threats and the high mortality rate of bacterial infections worldwide. Raman spectroscopy has recently gained popularity as an attractive robust approach for the molecular characterization, rapid identification, and accurate classification of a wide range of bacteria. In this dissertation, Raman spectroscopy utilizing advanced statistical techniques was used to identify and discriminate between different pathogenic and non-pathogenic bacterial strains of E. coli and Staphylococcus aureus bacterial species by probing the molecular compositions of the cells. The five-carbon sugar xylitol, which cannot be metabolized by the oral and nasopharyngeal bacteria, had been recognized by clinicians as a preventive agents for dental caries and many studies have demonstrated that xylitol causes a reduction in otitis media (chronic inner ear infections) and other nasopharyngeal infections. Raman spectroscopy was used to characterize the uptake and metabolic activity of xylitol in pathogenic (viridans group Streptococcus) and nonpathogenic (E. coli) bacteria by taking their Raman spectra before xylitol exposure and after growing with xylitol and quantifying the significant differences in the molecular vibrational modes due to this exposure. The results of this study showed significant stable spectral changes in the S. viridians bacteria induced by xylitol and those changes were not the same as in some E. coli strains. Finally, Raman spectroscopy experiments were conducted to provide important information about the function of a certain protein (wag 31) of Mycobacterium tuberculosis using a relative non-pathogenic bacterium called Mycobacterium smegmatis. Raman spectra of conditional mutants of bacteria expressing three different phosphorylation forms of wag31 were collected and analyzed. The results show that that the phosphorylation of wag31 causes significant differences in the molecular structure, namely the quantity of amino acids associated with peptidoglycan precursor proteins and lipid II as observed in the Raman spectra of these cells. Raman spectra were also acquired from the isolated cell envelope fraction of the cells expressing different forms of wag31 and the results showed that a significant number of the molecular vibrational differences observed in the cells was also observed in the cell envelope fraction, indicating that these differences are localized in the cell envelope

A Comparative Study between Jordanian Overall Heat Transfer Coefficient (U-Value) and International Building Codes, With Thermal Bridges Effect Investigation

Depletion of fossil fuel and the environmental effect associated with the use of it have made the topic of “thermal insulation regulations” a major concern in country Jordan and worldwide. This paper reviews the overall heat transfer coefficient U-value in Jordanian code for the building envelope, which represents how much the building envelope transfer heat to the outside environment. U-value was reviewed with respect to the following factors, heating degree days, the heating load required to achieve thermal comfort. Based on the review a new U-value of 0.65 W/m2.K was proposed and it was found that this value reduces the energy demand almost 50%. Moreover, the thermal bridge effect was investigated and it was found that an obvious increase in the U-value is present when having thermal bridges; this will affect the energy demand, almost 200%

Multipole (E1, M1, E2, M2, E3, M3) transition wavelengths and rates between 3l5l' excited and ground states in nickel-like ions

A relativistic many-body method is developed to calculate energy and transition rates for multipole transitions in many-electron ions. This method is based on relativistic many-body perturbation theory (RMBPT), agrees with MCDF calculations in lowest-order, includes all second-order correlation corrections and includes corrections from negative energy states. Reduced matrix elements, oscillator strengths, and transition rates are calculated for electric-multipole (dipole (E1), quadrupole (E2), and octupole (E3)) and magnetic-multipole (dipole (M1), quadrupole (M2), and octupole (M3)) transitions between 3l5l' excited and ground states in Ni-like ions with nuclear charges ranging from Z = 30 to 100. The calculations start from a 1s22s22p63s23p63d10} Dirac-Fock potential. First-order perturbation theory is used to obtain intermediate-coupling coefficients, and second-order RMBPT is used to determine the matrix elements. A detailed discussion of the various contributions to the dipole matrix elements and energy levels is given for nickellike tungsten (Z = 74). The contributions from negative-energy states are included in the second-order E1, M1, E2 M2, E3, and M3 matrix elements. The resulting transition energies and transition rates are compared with experimental values and with results from other recent calculations. These atomic data are important in modeling of M-shell radiation spectra of heavy ions generated in electron beam ion trap experiments and in M-shell diagnostics of plasmas.Comment: 21 pages, 8 figures, 11 table

Regulation of polar peptidoglycan biosynthesis by Wag31 phosphorylation in mycobacteria

Background. Sensing and responding to environmental changes is a central aspect of cell division regulation. Mycobacterium tuberculosis contains eleven Ser/Thr kinases, two of which, PknA and PknB, are key signaling molecules that regulate cell division/morphology. One substrate of these kinases is Wag31, and we previously showed that partial depletion of Wag31 caused morphological changes indicative of cell wall defects, and that the phosphorylation state of Wag31 affected cell growth in mycobacteria. In the present study, we further characterized the role of the Wag31 phosphorylation in polar peptidoglycan biosynthesis. Results. We demonstrate that the differential growth among cells expressing different wag31 alleles (wild-type, phosphoablative, or phosphomimetic) is caused by, at least in part, dissimilar nascent peptidoglycan biosynthesis. The phosphorylation state of Wag31 is found to be important for protein-protein interactions between the Wag31 molecules, and thus, for its polar localization. Consistent with these results, cells expressing a phosphomimetic wag31 allele have a higher enzymatic activity in the peptidoglycan biosynthetic pathway. Conclusions. The Wag31 Mtbphosphorylation is a novel molecular mechanism by which Wag31 Mtbregulates peptidoglycan synthesis and thus, optimal growth in mycobacteria. © 2010 Jani et al; licensee BioMed Central Ltd

Sensitive and specific discrimination of pathogenic and nonpathogenic Escherichia coli using Raman spectroscopy - a comparison of two multivariate analysis techniques

The determination of bacterial identity at the strain level is still a complex and time-consuming endeavor. In this study, visible wavelength spontaneous Raman spectroscopy has been used for the discrimination of four closely related Escherichia coli strains: pathogenic enterohemorrhagic E. coli O157:H7 and non-pathogenic E. coli C, E. coli Hfr K-12, and E. coli HF4714. Raman spectra from 600 to 2000 cm-1 were analyzed with two multivariate chemometric techniques, principal component-discriminant function analysis and partial least squares-discriminant analysis, to determine optimal parameters for the discrimination of pathogenic E. coli from the non-pathogenic strains. Spectral preprocessing techniques such as smoothing with windows of various sizes and differentiation were investigated. The sensitivity and specificity of both techniques was in excess of 95%, determined by external testing of the chemometric models. This study suggests that spontaneous Raman spectroscopy with visible wavelength excitation is potentially useful for the rapid identification and classification of clinically-relevant bacteria at the strain level

Influence of Storage Temperature and Duration of Tomato Leaf Samples on Proline Content

In arid and semi-arid countries such as Jordan, shortage in water sources might affect agricultural development and reduces the effectiveness of economic benefits of most crops planted in such areas. Tomato is an important agricultural crop and faces severe drought stress due to climate changes, therefore, measurement of proline accumulation in plant tissues is used as an indicator for drought stress tolerance. This research was conducted at Jarash University Campus in northern Jordan. A field experiment was carried out to investigate the impact of different storage temperature (+4ºC, - 20ºC and -80ºC) and different storage durations (0, 3, 6 and 11 weeks) on proline content in five different Jordanian tomato landraces. Results indicated that the average free proline content for samples tested directly after leaves collection was 7.1 µmol/g. Proline content in leaves stored at +4 ºC for 3, 6, and 11 weeks was 4.8, 1.8, and 1.1µmol/g, respectively, while for -20ºC was 11.8, 7.9, and 9.5 µmol/g for samples stored for 3, 6, 11 weeks respectively. In contrast the highest values for these parameters were obtained from samples stored at -80ºC, the average measured values of free proline content were 9.5, 7.8, and 12.9 µmol/g at 3, 6, and 11 weeks of storage, respectively. Based on the results obtained by this research, it is recommended to measure proline content directly after leaves collection. However, for large number of samples, keeping the samples at -20ºC not longer than six weeks could be a solution. Finally, we highly recommend the development of in-field method for measurement of free proline content

Regulation of Polar Peptidoglycan Biosynthesis by Wag31 Phosphorylation in Mycobacteria

<p>Abstract</p> <p>Background</p> <p>Sensing and responding to environmental changes is a central aspect of cell division regulation. <it>Mycobacterium tuberculosis </it>contains eleven Ser/Thr kinases, two of which, PknA and PknB, are key signaling molecules that regulate cell division/morphology. One substrate of these kinases is Wag31, and we previously showed that partial depletion of Wag31 caused morphological changes indicative of cell wall defects, and that the phosphorylation state of Wag31 affected cell growth in mycobacteria. In the present study, we further characterized the role of the Wag31 phosphorylation in polar peptidoglycan biosynthesis.</p> <p>Results</p> <p>We demonstrate that the differential growth among cells expressing different <it>wag31 </it>alleles (wild-type, phosphoablative, or phosphomimetic) is caused by, at least in part, dissimilar nascent peptidoglycan biosynthesis. The phosphorylation state of Wag31 is found to be important for protein-protein interactions between the Wag31 molecules, and thus, for its polar localization. Consistent with these results, cells expressing a phosphomimetic <it>wag31 </it>allele have a higher enzymatic activity in the peptidoglycan biosynthetic pathway.</p> <p>Conclusions</p> <p>The Wag31_Mtbphosphorylation is a novel molecular mechanism by which Wag31_Mtbregulates peptidoglycan synthesis and thus, optimal growth in mycobacteria.</p