The cell envelope of mycobacteria is critical for the survival and virulence of pathogenic species during infection, and its biosynthesis has been a proven drug target. Therefore, finding new targets in the biosynthetic pathway of cell envelope components is of great interest. Mycobacterium smegmatis is a model organism for the study of the devastating pathogen Mycobacterium tuberculosis. Previously, lipomannan elongation factor A (LmeA) has been identified as a cell envelope protein that is critical for the control of mannan chain length of lipomannan (LM) and lipoarabinomannan (LAM), lipoglycan components of the cell envelope. The deletion mutant, ∆lmeA, accumulates abnormal LM/LAM with fewer mannan residues. To understand the importance of this protein, the antibiotic sensitivity of ∆lmeA was tested using a resazurin-based viability assay. We found that the lmeA deletion leads to increased sensitivities to antibiotics such as vancomycin and erythromycin, and lmeA overexpression leads to increased antibiotic resistance. To directly test if the increased antibiotic sensitivity is due to the defective permeability barrier, we used an ethidium bromide uptake assay and found that ∆lmeA is more efficient in taking up ethidium bromide in the cell. We have also found that LmeA is important for protein stabilization under stress conditions. MptA is an α1,6-mannosyltransferase involved in elongation of LM and LAM mannan chain. During stress conditions in the ΔlmeA mutant, levels of MptA decrease significantly relative to wild-type. This also results in delayed doubling time after stress, a phenotype not seen in this mutant under normal growth conditions. In addition, the ΔlmeA mutant has differential protein expression during stress conditions relative to ΔlmeA in log phase, or to wild-type in either condition. To help elucidate the role of LmeA at the molecular level, binding behavior of this protein to membrane fractions was determined. In a subcellular fractionation analysis, LmeA localizes to fractions containing plasma membrane, which is tightly bound to cell wall layers. To test the binding of LmeA to membrane further, LmeA was heterologously expressed in Escherichia coli, purified, and mixed M. smegmatis cell lysate. LmeA localized to intracellular domain fractions (IMD), indicating that LmeA is capable of localizing to fractions containing only plasma membrane. Consistent with this finding, LmeA is capable of binding to spheroplasts in both an ELISA setting as well as in a sucrose gradient fractionation setting. It has also been determined that ΔlmeA has a defective capsular layer with a unique phenotype relative to other strains. We have concluded that LmeA is important for antibiotic resistance, cell envelope permeability, capsule formation, stress response, and have also determined its binding properties.

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Les entérocoques, sont des bactéries commensales du tube digestif de l’homme et des animaux, mais certaines espèces, comme Enterococcus faecium, sont aussi des pathogènes opportunistes majeurs souvent multi-résistants aux antibiotiques. Nous avons étudié l’impact de molécules non antibiotiques utilisées dans les unités de soins intensifs, sur la virulence et la résistance d’une souche clinique de E. faecium par une approche microscopique, une analyse du peptidoglycane et une analyse trancriptomique. Ce travail nous a permis de décrire l’effet antibactérien de la caspofungine, molécule antifongique. Nous avons également étudié deux nouveaux mécanismes de résistance chez E. faecium i) la résistance aux lincosamides, streptogramines A et pleuromutilines (phénotype LSAP) par la mutation ponctuelle d’un gène codant pour une protéine ABC de type II. ii) la diminution de sensibilité à la tigécycline due à l’apparition de mutations au sein du gène rpsJ codant pour la protéine ribosomale S10 jouant un rôle dans la formation de la sous-unité 30S du ribosome. Enfin, nous avons participé à une étude sur Enterococcus hirae, espèce qui induirait la production de sous-populations de lymphocytes T permettant d’augmenter l’efficacité in vivo du cyclophosphamide (CTX) dans le traitement de tumeurs chez la souris. Une caractérisation des facteurs de virulence, de la résistance aux antibiotiques et du pouvoir de colonisation d’une collection de souches d’E. hirae a été menée, de même qu’une étude transcriptomique en présence de CTX et une étude de génomique comparative, afin de caractériser cette espèce dans l’optique de son utilisation comme oncobiotique. / Enterococci are commensal bacteria of the human and animal gastrointestinal tract, but some species as Enterococcus faecium, are also major opportunistic pathogens often multiply resistant to antibiotics. We studied the impact of non-antibiotic molecules widely used in intensive care units on fitness, virulence and resistance of a clinical isolate of E. faecium belonging to CC17 by a microscopic approach, a peptidoglycan analysis and a trancriptomic analysis. This work allowed us to demonstratethe antimicrobial effect of caspofungin, molecule known for its antifungal activity. We also characterized two novel resistance mechanisms found in E. faecium: i) resistance to lincosamides, streptogramines A and pleuromutilins (LSAP phenotype) linked to a point mutation in a gene encoding for a type-II ABC protein. ii) decreased susceptibility to tigecycline due to the occurrence of mutations within the rpsJ gene encoding the S10 ribosomal protein that plays a role in 30S ribosomal subunit formation. Finally, we participated to a study concerning Enterococcus hirae, species that induces the production of sub-populations of T lymphocytes that increase the in vivo efficacy of cyclophosphamide (CTX) in the treatment of murine tumors. A characterization of the virulence factors, antibiotic resistance profiles and colonization capacities of a collection of E. hirae isolates was carried out. A transcriptomic study in the presence of CTX and a comparative genomic study were also done, in order to characterize this species in view of its use as an oncobiotic.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / The incidence of multiple primary neoplasms has been increasing over the years. Within this group, the coexistence of primary prostate cancer and primary colorectal cancer is one of the most frequent. The objective of this case report is to present the case of a 76-year-old male patient who presented the diagnosis of prostate cancer and synchronous rectal cancer. To this end, his clinical history in the oncological service of the Hospital Militar Central del Perú (tertiary hospital) has been reviewed. / Revisión por pares

Mycobacterium tuberculosis (Mtb) remains a global challenge that has been worsened by the emergence of drug resistant strains of Mtb. We used publicly available Next Generation Sequencing (NGS) and drug susceptibility (DST) data to develop “Resistance sniffer”, an online software program for the rapid prediction of lineage and Mtb drug resistance. Based on the distribution of polymorphisms in the genomes of Mtb, we calculated the power of association between the polymorphisms in different clades of Mtb and resistance to 13 anti-TB drugs. Our data suggests that the development of drug resistance in Mtb is a stepwise process that involves the accumulation of polymorphisms in the Mtb genome. We carefully curated the polymorphisms based on their association powers to create a diagnostic key that captures patterns of these polymorphisms that can be used to predict lineage and drug resistance in Mtb. This diagnosis key was incorporated into the Resistance Sniffer tool, an online software program that we developed for the rapid diagnosis of drug resistance in Mtb. The tool was tested using sequence data from the South Africa Medical Research Council (SA-MRC). Our data suggests that the majority of the strains in SA may have been brought by the arrival of European settlers while the more resistant strains may have been introduced in the region by Asian travellers later on. We next sought to determine non-random associations between polymorphic sites in genomes of Mtb. Using the attributable risk (Ra) statistical methods, we distinguished between functional associations and associations that may have been due to genetic drift events for different Mtb clades. We then integrated the (Ra) data with drug susceptibility and annotation data to generate networks in Cytoscape 3.71. These networks were then used to infer evolutionary trajectories that drive the emergence and fixation of the drug resistant phenotype in different clades of Mtb. We demonstrate that strains from the Lineage 1.2 are associated with less complex functional associations compared to the strains from other clades such as the Asian and Euro-American clades. Our data also shows that the predisposition of strains from the Asian clades to develop multi-drug resistance may be attributed to a complex network of functional interactions of mutations in genes that are involved in several aspects of Mtb physiology such as cell wall modelling, lipid metabolism, stress response and DNA repair. / Dissertation (MSc)--University of Pretoria, 2019. / Biochemistry / MSc / Unrestricted

The existence of pharmaceuticals in the environment has some adverse effects, and may pose threat to the organisms in the environment. The possibility of removing certain pharmaceuticals from wastewater was tested using Octolig®, a commercially available material with polyethyldiamine moieties covalently attached to high-surface area silica gel. Selected drug models were subjected to column chromatography in efforts to effect removal by means of ion encapsulation, the effectiveness of which would depend upon having appropriate anionic functional groups. The experimental results suggested that the model compounds, Rose Bengal, Eosin Y, Erythrosine , ZPS, and Lissamine Green B were successfully encapsulated by Octolig®, while Methylene Blue with quaternary ammonium groups was (statistically) not. In contrast, complete success was attained for removing of each of three xanthenylbenzenes (Rose Bengal, Eosin Y, Erythrosine) that have both phenolic and carboxylic acid groups. In addition complete success was attained for ZPS (zinc phthalocyaninetetrasulfonate) with sulfonate groups present. A test of a real pharmaceutical compound, Amoxicillin, indicated that Octolig® can be used to remove this compound from aqueous media.

The overuse of antibiotics is a large problem in healthcare today, accelerating the development of microbial resistance to antibiotics. Antibiotic stewardship campaigns have been implemented to help clinicians curb their use. Procalcitonin is a serum peptide and marker of inflammation secreted in response to microbial toxins. For this reason it is more specific to bacterial infections than other markers of general inflammation , like Creactive protein. The population of patients with sepsis in the Intensive Care Unit is one in which extended durations of antibiotics are used. The FDA has approved use of procalcitonin to guide de-escalation of antibiotic therapy in critically ill patients with sepsis to avoid both antibiotic overuse and antibiotic related side effects. Review of current literature shows that procalcitonin is efficacious in reducing duration of antibiotic therapy in patients with sepsis in the ICU setting. This result, however, is not being observed in clinical practice. This discrepancy is due to the inappropriate use of procalcitonin that does not align with use outlined in randomized control trials. We propose a study to determine how procalcitonin is being used in clinical practice in four Boston area hospital Intensive Care Units. Through chart review, we will identify patients in the Intensive Care Unit with sepsis from 2013-2018 recording patient demographic information and patient characteristics. We will determine whether they had PCT measured during their stay, and if they did, whether or not discontinuation of antibiotics was in accordance with FDA’s proposed algorithm. We will aim to compare whether discontinuing antibiotic therapy in accordance with the FDA’s procalcitonin deescalation algorithm is associated with reduced duration of antibiotic therapy or incidence of Clostridium Difficile infection. In conducting this study, we hope to identify patterns of procalcitonin use in clinical practice and provide further evidence that using the algorithm to guide therapy can serve as an effective tool in reducing exposure to unnecessary antibiotics and the complications from their use.

Objective: To determine whether echocardiography affects the decisions to use beta blockers or to recommend bacterial endocarditis prophylaxis in patients suspected of having mitral valve prolapse (MVP). Design: Retrospective review of echocardiograms and clinical records. Setting: Military tertiary care hospital. Patients: 127 patients with clinically suspected MVP (105) or incidentally discovered MVP (22). Main results: Beta blockers were used more often in patients with suspected MVP and positive echocardiograms (45%) than in patients with normal echocardiograms (13%, p<0.001). Bacterial endocarditis prophylaxis was recommended more often in patients with suspected MVP and positive echocardiograms (65%) than in patients with normal echocardiograms (11%, p<0.001). Presence or absence of a murmur did not influence the decision to recommend bacterial endocarditis prophylaxis. Patients in whom MVP was incidentally discovered were unlikely to receive either beta blockers or the recommendation for bacterial endocarditis prophylaxis. Conclusions: The results of echocardiography affect the decisions to use beta blockers or to recommend bacterial endocarditis prophylaxis in patients with suspected MVP.

Objectives: To compare and contrast the geographic and demographic distribution of bro β-lactamase and antibiotic MIC50/90 for 1440 global Moraxella catarrhalis isolates obtained from children and adults between 2001 and 2002. Methods: One thousand four hundred and forty M. catarrhalis isolates originating from seven world regions were investigated. The isolates were recovered from 411 children <5 years of age and 1029 adults >20 years of age. PCR-restriction fragment length polymorphism (RFLP) was performed to determine bro prevalence and to distinguish between bro types. MIC values of 12 different antibiotics were determined using the CLSI (formerly NCCLS) broth microdilution method. Results: Of the 1440 isolates, 1313 (91%) possessed the bro-1 gene and 64 (4%) possessed the bro-2 gene. Additionally, the prevalence of bro positivity between the child and adult age groups was significantly different (P<0.0001), though bro-1 and bro-2 prevalences within age groups were not significantly different. Consistently higher β-lactam MICs were observed for M. catarrhalis isolates originating in the Far East. Significant correlations in MICs were observed for several antibiotic combinations, including all five β-lactams with each other, and among the two quinolones. Conclusions: The worldwide prevalence of bro gene carriage in clinical isolates of M. catarrhalis is now approaching 95%, with children significantly more likely to harbour bro-positive isolates than adults. Further, statistically significant differences in the distribution of β-lactam MICs were observed between different world regions, particularly with respect to the Far East.