Phage infection, transfection and transformation of \u3ci\u3eMycobacterium avium\u3c/i\u3e complex and \u3ci\u3eMycobacterium paratuberculosis\u3c/i\u3e

Mycobacterium avium complex strains and Mycobacterium paratuberculosis are closely related intracellular pathogens affecting humans and animals. M. avium complex infections are a leading cause of morbidity and mortality in AIDS patients, and M. paratuberculosis is the agent of Johne\u27s disease in ruminants. Genetic manipulation of these micro-organisms would facilitate the understanding of their pathogenesis, the construction of attenuated vaccine strains and the development of new drugs and treatment methods. This paper describes the replication of mycobacterial shuttle phasmids and plasmids, and the expression of the firefly luciferase reporter gene in M. avium complex and M. paratuberculosis. The mycobacteriophage TM4 propagated on M. smegmatis or M. paratuberculosis plaqued at the same efficiency on these two mycobacterial hosts. Screening of M. avium complex and M. paratuberculosis clinical isolates with TM4-derived luciferase reporter phages demonstrated that the majority of these isolates were susceptible to TM4. Conditions for introduction of DNA were determined by transfection of M. paratuberculosis with TM4 DNA and applied to isolate kanamycin-resistant transformants of M. avium complex and M. paratuberculosis with Escherichia coli-Mycobacterium shuttle plasmids. Recombinant plasmids were recovered from transformants without apparent loss of DNA sequences. These results provide the basis for the genetic manipulation of these pathogenic mycobacterial species

Flux Balance Analysis with Objective Function Defined by Proteomics Data-Metabolism of Mycobacterium tuberculosis Exposed to Mefloquine

We present a study of the metabolism of the Mycobacterium tuberculosis after exposure to antibiotics using proteomics data and flux balance analysis (FBA). The use of FBA to study prokaryotic organisms is well-established and allows insights into the metabolic pathways chosen by the organisms under different environmental conditions. To apply FBA a specific objective function must be selected that represents the metabolic goal of the organism. FBA estimates the metabolism of the cell by linear programming constrained by the stoichiometry of the reactions in an in silico metabolic model of the organism. It is assumed that the metabolism of the organism works towards the specified objective function. A common objective is the maximization of biomass. However, this goal is not suitable for situations when the bacterium is exposed to antibiotics, as the goal of organisms in these cases is survival and not necessarily optimal growth. In this paper we propose a new approach for defining the FBA objective function in studies when the bacterium is under stress. The function is defined based on protein expression data. The proposed methodology is applied to the case when the bacterium is exposed to the drug mefloquine, but can be easily extended to other organisms, conditions or drugs. We compare our method with an alternative method that uses experimental data for adjusting flux constraints. We perform comparisons in terms of essential enzymes and agreement using enzyme abundances. Results indicate that using proteomics data to define FBA objective functions yields less essential reactions with zero flux and lower error rates in prediction accuracy. With flux variability analysis we observe that overall variability due to alternate optima is reduced with the incorporation of proteomics data. We believe that incorporating proteomics data in the objective function used in FBA may help obtain metabolic flux representations that better support experimentally observed features

Evaluation of eight live attenuated vaccine candidates for protection against challenge with virulent Mycobacterium avium subspecies paratuberclosis in mice

Johne\u27s disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), which results in serious economic losses worldwide in farmed livestock such as cattle, sheep, and goats. To control this disease, an effective vaccine with minimal adverse effects is needed. In order to identify a live vaccine for Johne\u27s disease, we evaluated eight attenuated mutant strains of MAP using a C57BL/6 mouse model. The persistaence of the vaccine candidates was measured at 6, 12, and 18 weeks post vaccination. Only strains 320, 321, and 329 colonized both the liver and spleens up until the 12-week time point. The remaining five mutants showed no survival in those tissues,indicating their complete attenuation in the mouse model. The candidate vaccine strains demonstrated different levels of protection based on colonization of the challenge strain in liver and spleen tissues at 12 and 18 weeks post vaccination. Based on total MAP burden in both tissues at both time points, strain 315 (MAP 1566::Tn 5370) was the most protective where as strain 318 (intergenic Tn 5367 insertion between MAP 0282c and MAP 0283c) had the most colonization. Mice vaccinated with an undiluted commercial vaccine preparation displayed the highest bacterial burden as well as enlarged spleens indicative of a strong infection. Selected vaccine strains that showed promise in the mouse model were moved forward in to a goat challenge model. The results suggest that the mouse trial, as conducted, may have a relatively poor predictive value for protection in a ruminant host such as goats

A rational framework for evaluating the next generation of vaccines against Mycobacterium avium subspecies paratuberculosis

Since the early 1980s, several investigations have focused on developing a vaccine against Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne\u27s disease in cattle and sheep. These studies used whole-cell inactived vaccines that have proven useful in limiting disease progression, but have not prevented infection. In contrast, modified live vaccines that invoke a Th1 type immune response, may improve protection against infection. Spurred by recent advances in the ability to create defined knockouts in MAP, several independent laboratories have developed modified live vaccine candidates by transcriptional mutation of virulence and metablolic genes in MAP. In order to accelerate the process of identification and comparative elvaluation of he most promising modified live MAP vaccine candidates, members of a multi-institutional USDA- funded research consortium, the Johne\u27s disease integrated program (JDIP), met to established a standardized testing platform using agreed upon protocols. A total of 22 candidates vaccine strains developed in five independent laboratories in the United States and New Zealand voluntarily entered into a double blind gated trial pipeline. In Phase I, the survival characteristics of each candidate were determined in bovine macrophages. Attenuated strains moved to Phase II, where tissue colonization of C57/BL6 mice were evaluated in a challenge model. In Phase III, five promising candidates from Phase I and II were evaluated for their ability to reduce fecal shedding, tissue colonization and pathology in a baby goat challenge model. Formation of a multi-institutional consortium for vaccine strain evaluation has revealed insights for the implementation of vaccine trials for Johne\u27s disease and other animals pathogens. We conclude by suggesting the best way forward based on this 3-phase trial experience and challenge the rationale for use of a macrophage-to-mouse-to native host pipeline for MAP vaccine development

Characterization of the inflammatory phenotype of Mycobacterium avium subspecies paratuberculosis using a novel cell culture passage model

Understanding the pathogenic mechanisms of Mycobacterium avium subspecies paratuberculosis (MAP) and the host responses to Johne's disease is complicated by the multi-faceted disease progression, late-onset host reaction and the lack of available ex vivo infection models. We describe a novel cell culture passage model that mimics the course of infection in vivo. The developed model simulates the interaction of MAP with the intestinal epithelial cells, followed by infection of macrophages and return to the intestinal epithelium. MAP internalization triggers a minimal inflammatory response. After passage through a macrophage phase, bacterial reinfection of MDBK epithelial cells, representing the late phase of intestinal mucosal infection, is associated with increased synthesis of the pro-inflammatory transcripts of IL-6, CCL5, IL-8 and IL-18, paired with decreased levels of TGFβ. Transcriptome analysis of MAP from each stage of epithelial cell infection identified increased expression of lipid biosynthesis and lipopeptide modification genes in the inflammatory phenotype of MAP. Total lipid analysis by HPLC-ES/MS indicates different lipidomic profiles between the two phenotypes and a unique set of lipids composing the inflammatory MAP phenotype. The presence of selected upregulated lipid-modification gene transcripts in samples of ileal tissue from cows diagnosed with Johne's disease supports and validates the model. By using the relatively simple cell culture passage model, we show that MAP alters its lipid composition during intracellular infection and acquires a pro-inflammatory phenotype, which likely is associated with the inflammatory phase of Johne's disease

Avaliação do Uso Profilático de Antibióticos em um Hospital de Câncer

O trabalho mostra e discute os padrões de uso de antibióticos de forma profilática em 6129 altas e óbitos durante 19 meses em um hospital de câncer. Foram encontradas altas freqüências de uso em cirurgias limpas. Os antibióticos mais utilizados foram as cefalosporinas, seguidas pelas benzil-penicilinas e cloranfenicol

Global economic burden of unmet surgical need for appendicitis

Background: There is a substantial gap in provision of adequate surgical care in many low-and middle-income countries. This study aimed to identify the economic burden of unmet surgical need for the common condition of appendicitis. Methods: Data on the incidence of appendicitis from 170 countries and two different approaches were used to estimate numbers of patients who do not receive surgery: as a fixed proportion of the total unmet surgical need per country (approach 1); and based on country income status (approach 2). Indirect costs with current levels of access and local quality, and those if quality were at the standards of high-income countries, were estimated. A human capital approach was applied, focusing on the economic burden resulting from premature death and absenteeism. Results: Excess mortality was 4185 per 100 000 cases of appendicitis using approach 1 and 3448 per 100 000 using approach 2. The economic burden of continuing current levels of access and local quality was US $92 492 million using approach 1 and$73 141 million using approach 2. The economic burden of not providing surgical care to the standards of high-income countries was $95 004 million using approach 1 and$75 666 million using approach 2. The largest share of these costs resulted from premature death (97.7 per cent) and lack of access (97.0 per cent) in contrast to lack of quality. Conclusion: For a comparatively non-complex emergency condition such as appendicitis, increasing access to care should be prioritized. Although improving quality of care should not be neglected, increasing provision of care at current standards could reduce societal costs substantially

Pooled analysis of WHO Surgical Safety Checklist use and mortality after emergency laparotomy

Background The World Health Organization (WHO) Surgical Safety Checklist has fostered safe practice for 10 years, yet its place in emergency surgery has not been assessed on a global scale. The aim of this study was to evaluate reported checklist use in emergency settings and examine the relationship with perioperative mortality in patients who had emergency laparotomy. Methods In two multinational cohort studies, adults undergoing emergency laparotomy were compared with those having elective gastrointestinal surgery. Relationships between reported checklist use and mortality were determined using multivariable logistic regression and bootstrapped simulation. Results Of 12 296 patients included from 76 countries, 4843 underwent emergency laparotomy. After adjusting for patient and disease factors, checklist use before emergency laparotomy was more common in countries with a high Human Development Index (HDI) (2455 of 2741, 89.6 per cent) compared with that in countries with a middle (753 of 1242, 60.6 per cent; odds ratio (OR) 0.17, 95 per cent c.i. 0.14 to 0.21, P <0001) or low (363 of 860, 422 per cent; OR 008, 007 to 010, P <0.001) HDI. Checklist use was less common in elective surgery than for emergency laparotomy in high-HDI countries (risk difference -94 (95 per cent c.i. -11.9 to -6.9) per cent; P <0001), but the relationship was reversed in low-HDI countries (+121 (+7.0 to +173) per cent; P <0001). In multivariable models, checklist use was associated with a lower 30-day perioperative mortality (OR 0.60, 0.50 to 073; P <0.001). The greatest absolute benefit was seen for emergency surgery in low- and middle-HDI countries. Conclusion Checklist use in emergency laparotomy was associated with a significantly lower perioperative mortality rate. Checklist use in low-HDI countries was half that in high-HDI countries.Peer reviewe