In vitro susceptibility of Staphylococcus aureus strains isolated from cows with subclinical mastitis to different antimicrobial agents

Sensitivity to commercial teat dips (nonoxinol-9 iodine complex and chlorhexidine digluconate) of 56 Staphylococcus (S.) aureus strains isolated from quarter milk samples of various German dairy herds treated with different teat dipping schemes was investigated in this study. The minimum inhibitory concentration was determined using a broth macrodilution method according to the German Veterinary Association guidelines. The main objective of the current study was to induce in vitro resistance induction of S. aureus to chemical disinfectants. Ten different strains were repeatedly passed ten times in growth media with sub-lethal concentrations of disinfectants. Nine strains showed a significant reduction in susceptibility to the nonoxinol-9 iodine complex but only one strain developed resistance to chlorhexidine digluconate. Stability of the acquired resistance was observed in all S. aureus strains adapted to the nonoxinol-9 iodine complex and chlorhexidine digluconate. In contrast, simultaneous resistance to different antibiotics was not observed in any of the ten investigated S. aureus strains. However, the isolates exhibited a high degree of resistance to penicillin G. Based on these results, resistance of S. aureus to chemical disinfectants may be more likely to develop if the chemicals are used at concentrations lower than that required for an optimal biocidal effect

STRUCTURAL PERFORMANCE FOR WIDE-SHALLOW BEAM IN SHEAR (COMPARATIVE STUDY)

Many floor systems contain wide shallow beams to carry floor loads and transfer it to columns. For example, in bridge construction, a system of wide shallow beams (WSBs) may provide a simple and economical system to transfer loads from the slab deck to columns. In many of these design situations, it is often advantageous to use different member widths to minimize reinforcement conflicts and reduce overall congestion. This geometrical peculiarity deserves some attention and caution while dealing with the behaviour at ultimate limit states for shear and flexure. Seven wide-shallow beams with edge columns specimens were used to investigate the effect of width to depth ratios (b/d) and column width to beam width ratios (c/b) on the performance of shear capacity. The performances were measured in terms of deflection, ultimate loads, crack patterns, web reinforcement strains, and shear strain distribution a cross width. The results revealed that, the contribution of concrete formals of the international codes must be recalibrated

Role of the Occluded Conformation in Bacterial Dihydrofolate Reductases

Dihydrofolate reductase (DHFR) from Escherichia coli (EcDHFR) adopts two major conformations, closed and occluded, and movement between these two conformations is important for progression through the catalytic cycle. DHFR from the cold-adapted organism Moritella profunda (MpDHFR) on the other hand is unable to form the two hydrogen bonds that stabilize the occluded conformation in EcDHFR and so remains in a closed conformation during catalysis. EcDHFR-S148P and MpDHFR-P150S were examined to explore the influence of the occluded conformation on catalysis by DHFR. Destabilization of the occluded conformation did not affect hydride transfer but altered the affinity for the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) and changed the rate-determining step of the catalytic cycle for EcDHFR-S148P. Even in the absence of an occluded conformation, MpDHFR follows a kinetic pathway similar to that of EcDHFR with product release being the rate-limiting step in the steady state at pH 7, suggesting that MpDHFR uses a different strategy to modify its affinity for NADP+. DHFRs from many organisms lack a hydrogen bond donor in the appropriate position and hence most likely do not form an occluded conformation. The link between conformational cycling between closed and occluded forms and progression through the catalytic cycle is specific to EcDHFR and not a general characteristic of prokaryotic DHFR catalysis

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State

The origin of substrate preference in promiscuous enzymes was investigated by enzyme isotope labelling of the alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH). At physiological temperature, protein dynamic coupling to the reaction coordinate was insignificant. However, the extent of dynamic coupling was highly substrate-dependent at lower temperatures. For benzyl alcohol, an enzyme isotope effect larger than unity was observed, whereas the enzyme isotope effect was close to unity for isopropanol. Frequency motion analysis on the transition states revealed that residues surrounding the active site undergo substantial displacement during catalysis for sterically bulky alcohols. BsADH prefers smaller substrates, which cause less protein friction along the reaction coordinate and reduced frequencies of dynamic recrossing. This hypothesis allows a prediction of the trend of enzyme isotope effects for a wide variety of substrates

Minimization of dynamic effects in the evolution of dihydrofolate reductase

Protein isotope labeling is a powerful technique to probe functionally important motions in enzyme catalysis and can be applied to investigate the conformational dynamics of proteins. Previous investigations have indicated that dynamic coupling is detrimental to catalysis by dihydrofolate reductase (DHFR) from the mesophile Escherichia coli (EcDHFR). Comparison of DHFRs from organisms adapted to survive at a wide range of temperatures suggests that dynamic coupling in DHFR catalysis has been minimized during evolution; it arises from reorganizational motions needed to facilitate charge transfer events. Contrary to the behaviour observed for the DHFR from the moderate thermophile Geobacillus stearothermophilus (BsDHFR), the chemical transformation catalyzed by the cold-adapted bacterium Moritella profunda (MpDHFR) is only weakly affected by protein isotope substitutions at low temperatures, but the isotopically substituted enzyme is a substantially inferior catalyst at higher, non-physiological temperatures. QM/MM studies revealed that this behaviour is caused by the enzyme’s structural sensitivity to temperature changes, which enhances unfavorable dynamic coupling at higher temperatures by promoting additional recrossing trajectories on the transition state dividing surface. We postulate that these motions are minimized by fine-tuning DHFR flexibility through optimization of the free energy surface of the reaction, such that a nearly static reaction-ready configuration with optimal electrostatic properties is maintained under physiological conditions

Protein motions and dynamic effects in enzyme catalysis

The role of protein motions in promoting the chemical step of enzyme catalysed reactions remains a subject of considerable debate. Here, a unified view of the role of protein dynamics in dihydrofolate reductase catalysis is described. Recently the role of such motions has been investigated by characterising the biophysical properties of isotopically substituted enzymes through a combination of experimental and computational analyses. Together with previous work, these results suggest that dynamic coupling to the chemical coordinate is detrimental to catalysis and may have been selected against during DHFR evolution. The full catalytic power of Nature's catalysts appears to depend on finely tuning protein motions in each step of the catalytic cycle

Evaluation of in vitro effect of Fosfomycin on resistant Gram-negative pathogens in urinary tract infection

Background:Urinary tract infection (UTI) is considered one of the most common infections occurring in different ages. The increasing emergence and rapid spread of multidrug-resistant (MDR) pathogens has led to reuse older antimicrobials like Fosfomycin. This study aimed to evaluate the activity of Fosfomycin on MDR pathogens beside its effect on biofilm formation. Methods: A total of 116  MDR Gram -negative isolates  from  ICU patients suffering from UTI has been included in this study. Standard microbiological tests were done to identify the isolates. Susceptibility to various antibiotics was detected by disk diffusion method. Phenotypic tests for determining variousβ-lactamases were done. Minimal inhibitory concentration (MIC) for Fosfomycin was detected by agar dilution method. Formation of biofilm by the isolates with and without adding Fosfomycin was assessed by microtiter plate method. Results: The most frequently isolated pathogen was E. coli (70/116); 60.3% followed by Klebsiella spp. (31/116); 26.7%. Fosfomycin showed a high level of inhibitory effect on most of tested isolates ; E. coli revealed low resistance rate of 4.2%,while Klebsiella spp < /em>, Pseudomonas aeruginosa and Acinetobacter baumani showed resistance rate of 16% ,36%), and 50%, respectively.  A total of 72 (62.1%) isolates was ESBL producers, of which 92% isolates were Fosfomycin - sensitive , while 25(22%) isolates were MBL-positive, of which 88% were sensitive to Fosfomycin. Eighty-seven (75%) isolates were biofilm producers. Fosfomycin inhibited biofilm formation in 67(77%) isolates. Conclusion: ESBL and MBL producing Gram negative urinary pathogens showed high sensitivity level to Fosfomycin. Also, Fosfomycin had good inhibitory effect on their biofilm formation

Dihydrofolate reductase and the physical basis of enzyme catalysis

Dihydrofolate reductase (DHFR) is the enzyme that catalyses the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) in the presence of the cofactor reduced nicotinamide adenine dinucleotide phosphate (NADPH). The DHFR catalysed reaction has often been used to study enzymatic tunnelling and the contribution of protein dynamics to catalysis. To gain a better understanding of such phenomena and to investigate the key elements of structural adaptation in DHFR, in this thesis the hydride transfer reaction of DHFR from Moritella profunda (MpDHFR), a cold adapted enzyme, was studied and compared to the mesophilic and extensively studied enzyme from Escherichia coli (EcDHFR) and the thermophilic enzyme from Thermotoga maritima (TmDHFR). Chapter 1 gives a brief introduction to the thesis. Description of the materials and methods used in evaluating this work is reported in Chapter 2. In Chapter 3, the steady state and pre-steady state temperature dependences of the kinetic isotope effect (KIE) for the MpDHFR catalysed reaction was elevated, compared to data obtained for the mesophilic and the thermophilic DHFR homologues and the results interpreted according to the environmentally coupled tunnelling model. The work presented in Chapters 4 and 5 has investigated the role of dynamics during catalysis by DHFR using site directed mutagenesis. In Chapter 4, mutations were created in the GH loop for both EcDHFR and MpDHFR to elucidate the role of the occluded conformation during catalysis by DHFR. In Chapter 5, different MpDHFR and EcDHFR variants in the highly mobile M20 loop were generated and their temperature dependences of KIE were studied in addition to studying the two variants MpDHFR-G123V and MpDHFR-D124N in the catalytically important FG loop. The results obtained suggest that MpDHFR does not undergo the dynamical loop movements that have been recognized previously for EcDHFR in spite of following the same catalytic cycle. Further findings were found which contradict the current models that relate protein dynamics to catalysis efficiency, thus modifying these models has become essential. Chapter 6 has focused on studying the effect of different denaturants/salt concentrations on MpDHFR chemical step. Finally, a summary of the work presented in this thesis and future guidelines are provided in Chapter 7

Antimicrobial activities and metabolites profiling of Heliotropium bacciferum Forssk. methanolic extract

In the present study, Heliotropium bacciferum Forssk. methanolic extract was tested for its antibacterial activities against Pectobacterium carotovorum, P. atrosepticum, Ralstonia solanacearum, and Streptomyces scabiei bacterial strains. The plant extract was also tested for its antifungal properties against strains of Fusarium oxysporum, Botrytis cinerea, and Rhizoctonia solani. The potent antibacterial activities were recorded against R. solanacearum after treatment with the extract at a concentration of 1000 µg/mL with an inhibition zone of 9.67 ±0.57 mm. The methanolic extract also showed promising antifungal effects against B. cinerea and F. oxysporum, with fungal growth inhibition percentages of 86.22 ±0.33% and 82.00 ±0.55%, respectively, which were higher than the standard antifungal drug. The plant extract’s HPLC analysis identified 12 phenolic compounds and 6 flavonoid compounds, with HPLC peaks matching the used standards. Gallic acid, chlorogenic acid, coffeic acid, ellagic acid, coumaric acid, syringic acid, methyl gallate, ferulic acid, vanillin, pyrocatechol, and cinnamic acid were the phenolic compounds that were identified. The flavonoid compounds found in the extracts were daidzein, naringenin, quercetin, kaempferol, hesperetin, apigenin, and rutin, arranged from high to low abundance. Furthermore, according to the GC-MS analysis, the most abundant compounds detected in the plant extract were n-hexadecanoic acid, 9,12-octadecadienoic acid (z,z), á-sitosterol, betulin, phorbol, cis-13-octadecenoic acid, and octadecanoic acid. These compounds in HPLC and GC-MS analyses were proven to have antibacterial as well as antifungal properties. Based on the obtained antimicrobial results, H. bacciferum methanolic extracts could be recommended as a promising antibacterial and antifungal agent, as well as a safe alternative to many antimicrobial pesticides for the environment and human health