Natural evolution and engineering of transketolase

Transketolase (TK) is an important metabolic enzyme in all organisms. The enantioselective carbon-carbon bond forming action of TK makes it significantly interesting for biocatalysis. TKs from different organisms exhibit varied substrate specificities mediated by a small number of differing residues, giving insight into a potential route for engineering new enzymes. TK uses Thiamine Pyrophosphate (TPP) as cofactor, as do many other evolutionarily related enzymes. In Chapter 3, a phylogenetic analysis of the catalytic domains of TPP-dependent enzymes enabled the assembly of the evolutionary history of this enzyme family. In Chapter 4, the evolution of the differing substrate specificities of Eschericia coli TK and Saccharomyces cerevisiae TK from their most recent common ancestor enzyme was analysed. A detailed phylogenetic analysis of TK was performed, yielding the amino acid sequences of the ancient TKs. TKs linking the common ancestor of E. coli and S.cerevisiae with extant E. coli TK were "resurrected" and assayed for the P-Hydroxypyruvate (P-HPA) + glycoaldehyde (GA) reaction. The common ancestor TK and E. coli TK were assayed for many reactions to define their substrate repertoires and to elucidate any evolutionary trends in substrate specificity. p-HPA is the ideal donor substrate for TK, since it yields CO2, making reactions irreversible. p-HPA is not readily available commercially and is very expensive. To be industrially viable, a cheaper donor is needed. Pyruvate is much cheaper than P-HPA yet TK has never been shown to use pyruvate as a donor. Chapter 5 describes a comparison of TK with the pyruvate utilising TPP-dependent enzymes DXPS and PDC, suggesting residues which may confer pyruvate usage. Mutants were generated and tested for activity with pyruvate. In Chapter 6, the non-catalytic C-terminal domain of TK (TKC domain) was examined. The function of the TKC domain is currently undefined. In this chapter the TKC domain is removed and it is shown that the TK enzyme activity is retained

Investigating the impact of poverty on colonization and infection with drug-resistant organisms in humans: a systematic review

Background Poverty increases the risk of contracting infectious diseases and therefore exposure to antibiotics. Yet there is lacking evidence on the relationship between income and non-income dimensions of poverty and antimicrobial resistance. Investigating such relationship would strengthen antimicrobial stewardship interventions. Methods A systematic review was conducted following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. PubMed, Ovid, MEDLINE, EMBASE, Scopus, CINAHL, PsychINFO, EBSCO, HMIC, and Web of Science databases were searched in October 2016. Prospective and retrospective studies reporting on income or non-income dimensions of poverty and their influence on colonisation or infection with antimicrobial-resistant organisms were retrieved. Study quality was assessed with the Integrated quality criteria for review of multiple study designs (ICROMS) tool. Results Nineteen articles were reviewed. Crowding and homelessness were associated with antimicrobial resistance in community and hospital patients. In high-income countries, low income was associated with Streptococcus pneumoniae and Acinetobacter baumannii resistance and a seven-fold higher infection rate. In low-income countries the findings on this relation were contradictory. Lack of education was linked to resistant S. pneumoniae and Escherichia coli. Two papers explored the relation between water and sanitation and antimicrobial resistance in low-income settings. Conclusions Despite methodological limitations, the results suggest that addressing social determinants of poverty worldwide remains a crucial yet neglected step towards preventing antimicrobial resistance

Nasal Carriage and Antimicrobial Susceptibility of Staphylococcus aureus in healthy preschool children in Ujjain, India

<p>Abstract</p> <p>Background</p> <p>There is increasing evidence that community acquired <it>S. aureus </it>infections are spreading among healthy children. Nasal colonization with <it>S. aureus </it>plays pivotal role in the increasing prevalence of resistant community acquired <it>S. aureus </it>infections worldwide. A regular surveillance system is important in ensuring quality of patient care. The aim of the study was to assess the prevalence of and the factors associated with nasal carriage of <it>S. aureus </it>and its antibiotic sensitivity pattern among healthy children in Ujjain, India.</p> <p>Methods</p> <p>A prospective study was done in paediatric outpatient clinics of R.D. Gardi medical college Ujjain, India. Healthy children from 1 month to 59 months of age were included. Information on previously known risk factors for nasal colonization was collected using a pre-tested questionnaire. Swabs from anterior nares were collected and transported in Amies transport media with charcoal and cultured on 5% sheep blood agar. Antibiotic sensitivity tests were performed using Kirby Bauer's disc diffusion method according to performance standards of Clinical and Laboratory Standard Institute guidelines.</p> <p>Results</p> <p>Of the 1,562 children from 1-month up-to five years of age included in the study 98 children tested positive for nasal carriage of <it>S. aureus</it>. The prevalence of nasal carriage of <it>S. aureus </it>was 6.3% (95% CI 5.1-7.5) out of which 16.3% (95% CI 8.9-23.8) were methicillin-resistant <it>S. aureus </it>(MRSA). The factors associated with nasal carriage were "child attending preschool" (OR 4.26, 95% CI 2.25-8.03; <it>P </it>= 0.007) or "school" (OR 3.02, 95% CI 1.27-7.18; <it>P </it>< 0.001) and "family size more than 10 members" (OR 2.76 95% CI 1.06-7.15; <it>P </it>= 0.03). The sensitivity pattern of isolated <it>S. aureus </it>showed resistance to commonly used oral antibiotics while resistance to glycopeptides was not noted.</p> <p>Conclusions</p> <p>We found a relatively low rate of nasal carriage of <it>S. aureus </it>in children below five years when compared to children of older age groups in India. Yet, prevalence of MRSA was relatively high.</p

Interventions to Influence Consulting and Antibiotic Use for Acute Respiratory Tract Infections in Children: A Systematic Review and Meta-Analysis

BACKGROUND: Respiratory tract infections (RTIs) are common in children and generally self-limiting, yet often result in consultations to primary care. Frequent consultations divert resources from care for potentially more serious conditions and increase the opportunity for antibiotic overuse. Overuse of antibiotics is associated with adverse effects and antimicrobial resistance, and has been shown to influence how patients seek care in ensuing illness episodes. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a systematic review and meta-analysis to assess the effectiveness of interventions directed towards parents or caregivers which were designed to influence consulting and antibiotic use for respiratory tract infections (RTIs) in children in primary care. Main outcomes were parental consulting rate, parental knowledge, and proportion of children subsequently consuming antibiotics. Of 5,714 references, 23 studies (representing 20 interventions) met inclusion criteria. Materials designed to engage children in addition to parents were effective in modifying parental knowledge and behaviour, resulting in reductions in consulting rates ranging from 13 to 40%. Providing parents with delayed prescriptions significantly decreased reported antibiotic use (Risk Ratio (RR) 0.46 (0.40, 0.54); moreover, a delayed or no prescribing approach did not diminish parental satisfaction. CONCLUSIONS: IN ORDER TO BE MOST EFFECTIVE, INTERVENTIONS TO INFLUENCE PARENTAL CONSULTING AND ANTIBIOTIC USE SHOULD: engage children, occur prior to an illness episode, employ delayed prescribing, and provide guidance on specific symptoms. These results support the wider implementation of interventions to reduce inappropriate antibiotic use in children

Expressed sequence tag analysis of khat (Catha edulis) provides a putative molecular biochemical basis for the biosynthesis of phenylpropylamino alkaloids

Khat (Catha edulis Forsk.) is a flowering perennial shrub cultivated for its neurostimulant properties resulting mainly from the occurrence of (S)-cathinone in young leaves. The biosynthesis of (S)-cathinone and the related phenylpropylamino alkaloids (1S,2S)-cathine and (1R,2S)-norephedrine is not well characterized in plants. We prepared a cDNA library from young khat leaves and sequenced 4,896 random clones, generating an expressed sequence tag (EST) library of 3,293 unigenes. Putative functions were assigned to > 98% of the ESTs, providing a key resource for gene discovery. Candidates potentially involved at various stages of phenylpropylamino alkaloid biosynthesis from L-phenylalanine to (1S,2S)-cathine were identified

Molecular imaging in oncology: the acceptance of PET/CT and the emergence of MR/PET imaging

In the last decade, PET-only systems have been phased out and replaced with PET-CT systems. This merger of a functional and anatomical imaging modality turned out to be extremely useful in clinical practice. Currently, PET-CT is a major diagnostic tool in oncology. At the dawn of the merger of MRI and PET, another breakthrough in clinical imaging is expected. The combination of these imaging modalities is challenging, but has particular features such as imaging biological processes at the same time in specific body locations

Modeling methicillin-resistant Staphylococcus aureus in hospitals: Transmission dynamics, antibiotic usage and its history

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is endemic in many hospital settings, posing substantial threats and economic burdens worldwide. METHODS: We propose mathematical models to investigate the transmission dynamics of MRSA and determine factors that influence the prevalence of MRSA infection when antibiotics are given to patients to treat or prevent infections with either MRSA itself or other bacterial pathogens. RESULTS: Our results suggest that: (i) MRSA always persists in the hospital when colonized and infected patients are admitted; (ii) the longer the duration of treatment of infected patients and the lower the probability of successful treatment will increase the prevalence of MRSA infection; (iii) the longer the duration of contamination of health care workers (HCWs) and the more their contacts with patients may increase the prevalence of MRSA infection; (iv) possible ways to control the prevalence of MRSA infection include treating patients with antibiotic history as quickly and efficiently as possible, screening and isolating colonized and infected patients at admission, and compliance with strict hand-washing rules by HCWs. CONCLUSION: Our modeling studies offer an approach to investigating MRSA infection in hospital settings and the impact of antibiotic history on the incidence of infection. Our findings suggest important influences on the prevalence of MRSA infection which may be useful in designing control policies

Knowledge and awareness of the general public and perception of pharmacists about antibiotic resistance

Background Antibiotic resistance (AR) continues to be a serious problem. Many factors contribute to AR, including inappropriate use of antibiotics, in which both healthcare professionals and patients play a contributing role. This study aimed to assess the awareness and knowledge of antibiotic usage and AR among the general public (in affluent and deprived areas) and community pharmacists' (CPs') in Greater London. Methods A cross-sectional survey involving members of the public was conducted between July 2014 and February 2015. Stage one involved members of the public (N = 384) residing in affluent areas of London. The second stage targeted public (N = 384) in deprived areas of London. In addition, CPs (N = 240) across the same areas were also surveyed. Data analysis was performed using Microsoft Excel and SPSS Software packages. Results Response rate: 36% (n = 139/384) and 57% (n = 220/384) and 25% (n = 60/240) of public residing in affluent areas, deprived areas and of CPs respectively was achieved. Definitive trends in knowledge of how antibiotics work could not be drawn to distinguish between affluent and deprived areas. However, public respondents residing in affluent areas possessed better understanding of AR and prudent use of antibiotics, and this was statistically significant in both cases (p < 0.05). Exposure to an antibiotic campaign (32% in affluent areas, 17% in deprived areas) did not raise public respondents' knowledge on AR and only partially raised their general knowledge on antibiotics usage. Only 20% of public residing in deprived areas received counselling from a CP, among them 74% had an antibiotic prescribed on at least one previous occasion. Those who received counselling displayed better knowledge about concordance/adherence with respect to antibiotic usage (p < 0.05) whereas exposure to an antibiotic campaign made no significant impact on knowledge about concordance/adherence. Conclusion The study highlights that there has been no change in the status quo with respect to awareness of antibiotic usage and AR even after the implementation of several awareness campaigns in England. Those who benefited from CP counselling showed a significant better knowledge towards prudent antibiotic usage which stresses the importance of CPs' counselling on antibiotic prescription