Engineering large functional plasmids for biosafety.

Large bacterial plasmid constructs (generally 25-100 kb, but can be greater), such as those engineered with DNA encoding specific functions such as protein secretion or specialized metabolism, can carry antibiotic resistance genes and/or conjugation systems that typically must be removed before use in medical or environmental settings due to biosafety concerns. However, a convenient in vivo recombineering approach for intact large plasmids to sequentially remove multiple different genes using non-antibiotic selection methods is not described in the literature to our knowledge. We developed strategies and reagents for convenient removal of antibiotic resistance markers and conjugation genes while retaining non-antibiotic-based plasmid selection to increase practical utility of large engineered plasmids. This approach utilizes targeted lambda Red recombination of PCR products encoding the trpE and asd genes and as well as FLP/FRT-mediated marker removal. This is particularly important given that use of restriction enzymes with plasmids of this size is extremely problematic and often not feasible. This report provides the first example of the trpE gene/tryptophan prototrophy being used for recombineering selection. We applied this strategy to the plasmids R995+SPI-1 and R995+SPI-2 which encode cloned type III secretion systems to allow protein secretion and substrate delivery to eukaryotic cells. The resulting constructs are functional, stably maintained under conditions where the original constructs are unstable, completely defective for conjugative transfer, and transferred via electroporation.

Cardiac complications of malnutrition in adolescent patients: A narrative review of contemporary literature.

Eating disorders are common. Between 1% and 2% of adolescent females and 0.5% of males suffer from anorexia nervosa, bulimia nervosa, and binge eating disorder. Although suicide represents nearly half of the mortality in patients with eating disorders, a majority of the remainder is cardiac arrest, likely secondary to cardiovascular complications of eating disorders such as bradycardia, hypotension, QT interval changes, structural heart disease, and pericardial effusion. Bradycardia is suspected to be secondary to increased vagal tone and is a common finding in patients admitted with disordered eating. Similarly, hypotension and orthostatic abnormalities are common complications due to atrophy of peripheral muscles. Descriptive studies report prolongation of the corrected QT interval (QTc) in these patients relative to controls, albeit within the normal reference range. Structural heart disease is also common, with left ventricular mass reported as lower than predicted in several studies compared to healthy controls. Pericardial effusion is also commonly described, although it is possible that this is underestimated, as not all patients with eating disorders undergo echocardiograms. Further, refeeding syndrome as a result of treatment of eating disorders carries its own cardiac risks. Cardiac complications of malnutrition are common but reversible with appropriate management and recovery. It is imperative that providers are aware of the epidemiology of these complications, as it is only with a high clinical suspicion that proper evaluation including a thorough history and physical examination, electrocardiogram, and when necessary echocardiogram can be performed.

A tale of caution: prolonged Bacillus clausii bacteraemia after probiotic use in an immunocompetent child.

INTRODUCTION: Bacillus clausii as a probiotic supplement is increasingly used in both adult and paediatric patient populations. There is limited awareness about potential adverse effects. CASE PRESENTATION: We report a case of prolonged (111 days) B. clausii bacteraemia after brief probiotic use in a 17-month-old immunocompetent child, without a definite focus of infection and in the absence of predisposing risk factors or underlying co-morbidities. We identified seven probiotic use-associated cases of prolonged B. clausii bacteraemia (mean duration [range] 64 days [14-93 days] where data were available) in the literature, all with underlying co-morbidities. CONCLUSION: B. clausii probiotic preparations may cause prolonged bacteraemia, rendering patients with underlying co-morbidities as well as those with unrecognized risk factors vulnerable for significant infectious complications.