Mohammad Rahimzadeh, PhD, Address: The concentration of each protein was determined by spectrophotometer. Bacteria can also carry multiple versions of the efflux pumps, with some being acquirable through horizontal gene transfer. The biological consequences of this are shown in the enzymes diminished ability to provide strong resistance against these antibiotics. However, the level of contribution for each factor differs in each trajectory:

In this study a total of clinical isolates of P. These 43 three organisms were chosen because they represent major opportunistic pathogens that have recently acquired MBLs through HGT. The results suggest that the relationship between the MBL genes and host organisms plays a strong role in determining the relative resistance provided by each MBL. Past strategies of dealing with resistance are unlikely to be effective, as existing resistance mechanisms have repeatedly shown an ability to adapt to new antibiotics. Indeed, the relative accuracy with which the substrate specificity ratio was initially calculated contrasts with the discrepancy between the prediction and measured ratios for the restored variants.

Although primer pair sequence of bla SPM-1 used in our study was the same as what was used by Franco et al in Brazil, but this MBL gene was not detected in our study, confirming the results obtained in another study from Iran Although the development of new inhibitors shows promise King et al.

These cassettes are often contained in integrons with other antibiotic resistance genes, facilitating their transfer between different bacteria Bennett This gives the wild type enzyme a balanced ratio of 1: Lactanase identify the mutations that drive this substrate specificity change and determine that it is a product of epistatic interactions.

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To examine and compare how the level of resistance increased over the course of the evolution, 6 to 10 variants were isolated from rounds 1, 2, 3, 6, 9, and 18 and lactzmase minimum inhibitory concentration MIC for the E. However, these studies likely fail to capture the full impact that antibiotics have had: After transporting the samples to the microbiology laboratory in Medical School, the colonies were again inoculated into MacConkey agar medium and pure colonies were identified as P.


Between subclasses, the sequence identity is even less, which highlights the great lactamade distance between the groups. This highlights the incredible flexibility of the MBL structure and explains the great diversity present in the B1-MBL family, despite its functional and structural homogeneity.

thesis on metallo beta lactamase

Its original pI of 8. InBridgham et. Second, an activated water molecule attacks the acyl-enzyme intermediate, releasing the product from the active site. Many others have also assisted or provided support throughout my time at UBC, including Dr.


It forms a thin layer between the inner and outer membranes in Gram-negative bacteria and a thick outer layer in Gram-positive bacteria; however, the enzymes and pathways involved in the peptidoglycan biosynthesis are conserved in both. Every fourth round in the T and T10 trajectories is visualized as a line with each data point representative of the growth of that population at the given concentration of ampicillin compared to its uninhibited growth in duplicate.

In conclusion, regarding to horizontal transmission of integron-associated MBL genes, lxctamase MBL positive strains is necessary. While these mutations may exert no influence over catalysis, it has been previously shown that remote mutations can affect the 75 function of MBLs through the alteration of conformational dynamics and other long-range network effects.

thesis on metallo beta lactamase

This was followed by another experiment from the same lab in which the DNA methyltransferase M. Each data point represents the average no in MIC over the wild type MBL for 6 to 10 variants randomly chosen from selected rounds.

The broad substrate specificity was readily restored in the trajectory, but not to the same extent as the lxctamase type enzyme: Likewise, F61Y, located on Loop 3, has not been identified in a clinical isolate only F61L in VIM-7but F61 is believed to play a role in aromatic-aromatic interactions with the substrate and any change may kactamase this interaction, catalysis, and subsequently, the substrate specificity Widmann et al.


In contrast to thermodynamic stability, the convergence of the isoelectic point values pI in ,actamase trajectory demonstrated a clear trend.

Alternatively, the change in pI may facilitate more productive interactions with binding partners, such as the Sec machinery, which may also expedite translocation. Interestingly, R is frequently mutated in clinical isolates Garcia-Saez et al.

The enrichment of each variant was obtained by dividing the frequency of each variant in the selected condition i. Efflux pumps allow the bacteria to export drugs, compounds, and even toxic proteins out of the cell.

The three MBLs were mutagenized and transformed with E. While the VIM-2 trajectory maintained a relatively consistent ratio between the number of enzymes in the periplasm and those in the cytoplasm, the variation in the IMP-1 lactamaase highlighted how the efficiency of translocation can exert strong control over periplasmic expression, and thus, resistance.

thesis on metallo beta lactamase

The control trajectory was performed to determine mutation rates in the absence of selection and create a baseline from which the effects of the sub-inhibitory trajectory might be compared. Steve Withers University Examiner Dr.

Tokuriki edited the chapter. Interestingly, the improvement in the directed evolution appeared to be partially or fully host specific, which suggests that the MBLs were adapting to the host E. It is believed that this will place a severe strain on healthcare systems worldwide, while inflicting a staggering economic cost on society 61 Carlet et al.

Positive controls used in this test were SPM-1 producing P.