Pediatric Ventilation Liberation: Bundled Extubation Readiness and Analgosedation Pathways Decrease Mechanical Ventilation Duration and Benzodiazepine Exposure.

BACKGROUND: Recent studies reported that children on mechanical ventilation who were managed with an analgosedation approach and standardized extubation readiness testing experienced better outcomes, including decreased delirium and invasive mechanical ventilation duration. METHODS: This was a quality improvement project in a 24-bed pediatric ICU within a single center, including subjects ≤ 18 years old who required invasive mechanical ventilation via an oral or nasal endotracheal tube. The aim was to decrease the invasive mechanical ventilation duration for all the subjects by 25% within 9 months through the development and implementation of bundled benzodiazepine-sparing analgosedation and extubation readiness testing clinical pathways. RESULTS: In the pre-implementation cohort, there were 274 encounters, with 253 (92.3%) that met inclusion for ending in an extubation attempt. In the implementation cohort, there were 367 encounters with 332 (90.5%) that ended in an extubation attempt. The mean invasive mechanical ventilation duration decreased by 23% (Pre 3.95 d vs Post 3.1 d; P = .039) after the implementation without a change in the mean pediatric ICU length of stay (Pre 7.5 d vs Post 6.5 d; P = .42). No difference in unplanned extubation (P > .99) or extubation failure rates (P = .67) were demonstrated. Sedation levels as evaluated by the mean State Behavioral Scale were similar (Pre -1.0 vs Post -1.1; P = .09). The median total benzodiazepine dose administered decreased by 75% (Pre 0.4 vs Post 0.1 mg/kg/ventilated day; P < .001). No difference in narcotic withdrawal (Pre 17.8% vs Post 16.4%; P = .65) or with delirium treatment (Pre 5.5% vs Post 8.7%; P = .14) was demonstrated. CONCLUSIONS: A multidisciplinary, bundled benzodiazepine-sparing analgosedation and extubation readiness testing approach resulted in a reduction in mechanical ventilation duration and benzodiazepine exposure without impacting key balancing measures. External validity needs to be evaluated in similar centers and consensus on best practices developed.

Challenging Convention: Daytime Versus Nighttime Extubation in the Pediatric ICU.

BACKGROUND: The majority of pediatric extubations occur during day shift hours. There is a time-dependent relationship between mechanical ventilation duration and complications. It is not known if extubation shift (day vs night) correlates with pediatric extubation outcomes. Pediatric ventilation duration may be unnecessarily prolonged if extubation is routinely delayed until day shift hours. METHODS: We hypothesized that extubation failure would not correlate with shift of extubation and that ventilation duration at first extubation and that length of stay in the pediatric ICU (PICU) would be shorter for children extubated at night. This was a retrospective cohort study within one tertiary care, 24-bed, academic PICU. RESULTS: 582 ventilation encounters were included, representing 517 unique subjects. Status epilepticus was a more common diagnosis among night shift extubations (P = .005), whereas surgical airway conditions were more common among day shift extubations (P = .02). Mechanical ventilation duration at first extubation (37.6 vs 62.5 h, P < .001) and length of stay in the PICU (2.8 vs 4.5 d, P < .001) were shorter for night shift extubations. The extubation failure rate was 10.3% for day shift and 8.1% for night shift (P = .40). Logistic regression modeling at the level of the unique subject indicated that extubation shift was not associated with extubation failure (P = .44). The majority of re-intubation events occurred on the shift opposite of extubation. There was no difference in complications according to shift of re-intubation (P = .72). CONCLUSIONS: Extubation failure was not independently associated with extubation shift in this single-center study. Ventilation liberation should be considered at the first opportunity dictated by clinical data and patient-specific factors rather than by the time of day at centers with similar resources.

Microbial gas vesicles as nanotechnology tools: exploiting intracellular organelles for translational utility in biotechnology, medicine and the environment.

A range of bacteria and archaea produce gas vesicles as a means to facilitate flotation. These gas vesicles have been purified from a number of species and their applications in biotechnology and medicine are reviewed here. Halobacterium sp. NRC-1 gas vesicles have been engineered to display antigens from eukaryotic, bacterial and viral pathogens. The ability of these recombinant nanoparticles to generate an immune response has been quantified both in vitro and in vivo. These gas vesicles, along with those purified from Anabaena flos-aquae and Bacillus megaterium, have been developed as an acoustic reporter system. This system utilizes the ability of gas vesicles to retain gas within a stable, rigid structure to produce contrast upon exposure to ultrasound. The susceptibility of gas vesicles to collapse when exposed to excess pressure has also been proposed as a biocontrol mechanism to disperse cyanobacterial blooms, providing an environmental function for these structures.

Agrichemicals and antibiotics in combination increase antibiotic resistance evolution.

Antibiotic resistance in our pathogens is medicine's climate change: caused by human activity, and resulting in more extreme outcomes. Resistance emerges in microbial populations when antibiotics act on phenotypic variance within the population. This can arise from either genotypic diversity (resulting from a mutation or horizontal gene transfer), or from differences in gene expression due to environmental variation, referred to as adaptive resistance. Adaptive changes can increase fitness allowing bacteria to survive at higher concentrations of antibiotics. They can also decrease fitness, potentially leading to selection for antibiotic resistance at lower concentrations. There are opportunities for other environmental stressors to promote antibiotic resistance in ways that are hard to predict using conventional assays. Exploiting our previous observation that commonly used herbicides can increase or decrease the minimum inhibitory concentration (MIC) of different antibiotics, we provide the first comprehensive test of the hypothesis that the rate of antibiotic resistance evolution under specified conditions can increase, regardless of whether a herbicide increases or decreases the antibiotic MIC. Short term evolution experiments were used for various herbicide and antibiotic combinations. We found conditions where acquired resistance arises more frequently regardless of whether the exogenous non-antibiotic agent increased or decreased antibiotic effectiveness. This is attributed to the effect of the herbicide on either MIC or the minimum selective concentration (MSC) of a paired antibiotic. The MSC is the lowest concentration of antibiotic at which the fitness of individuals varies because of the antibiotic, and is lower than MIC. Our results suggest that additional environmental factors influencing competition between bacteria could enhance the ability of antibiotics to select antibiotic resistance. Our work demonstrates that bacteria may acquire antibiotic resistance in the environment at rates substantially faster than predicted from laboratory conditions.

Herbicide ingredients change Salmonella enterica sv. Typhimurium and Escherichia coli antibiotic responses.

Herbicides are frequently released into both rural and urban environments. Commercial herbicide formulations induce adaptive changes in the way bacteria respond to antibiotics. Salmonella enterica sv. Typhimurium and Escherichia coli were exposed to common co-formulants of formulations, and S. enterica sv. Typhimurium was exposed to active ingredients dicamba, 2,4-D and glyphosate to determine what ingredients of the commercial formulations caused this effect. Co-formulants Tween80 and carboxymethyl cellulose induced changes in response, but the pattern of the responses differed from the active ingredients, and effect sizes were smaller. A commercial wetting agent did not affect antibiotic responses. Active ingredients induced changes in antibiotic responses similar to those caused by complete formulations. This occurred at or below recommended application concentrations. Targeted deletion of efflux pump genes largely neutralized the adaptive response in the cases of increased survival in antibiotics, indicating that the biochemistry of induced resistance was the same for formulations and specific ingredients. We found that glyphosate, dicamba, and 2,4-D, as well as co-formulants in commercial herbicides, induced a change in susceptibility of the potentially pathogenic bacteria E. coli and S. enterica to multiple antibiotics. This was measured using the efficiency of plating (EOP), the relative survival of the bacteria when exposed to herbicide and antibiotic, or just antibiotic, compared to survival on permissive media. This work will help to inform the use of non-medicinal chemical agents that induce changes in antibiotic responses.

The Aspergillus fumigatus farnesyltransferase ß-subunit, RamA, mediates growth, virulence, and antifungal susceptibility.

Post-translational prenylation mechanisms, including farnesylation and geranylgeranylation, mediate both subcellular localization and protein-protein interaction in eukaryotes. The prenyltransferase complex is an αß heterodimer in which the essential α-subunit is common to both the farnesyltransferase and the geranylgeranyltransferase type-I enzymes. The ß-subunit is unique to each enzyme. Farnesyltransferase activity is an important mediator of protein localization and subsequent signaling for multiple proteins, including Ras GTPases. Here, we examined the importance of protein farnesylation in the opportunistic fungal pathogen Aspergillus fumigatus through generation of a mutant lacking the farnesyltransferase ß-subunit, ramA. Although farnesyltransferase activity was found to be non-essential in A. fumigatus, diminished hyphal outgrowth, delayed polarization kinetics, decreased conidial viability, and irregular distribution of nuclei during polarized growth were noted upon ramA deletion (ΔramA). Although predicted to be a target of the farnesyltransferase enzyme complex, we found that localization of the major A. fumigatus Ras GTPase protein, RasA, was only partially regulated by farnesyltransferase activity. Furthermore, the farnesyltransferase-deficient mutant exhibited attenuated virulence in a murine model of invasive aspergillosis, characterized by decreased tissue invasion and development of large, swollen hyphae in vivo. However, loss of ramA also led to a Cyp51A/B-independent increase in resistance to triazole antifungal drugs. Our findings indicate that protein farnesylation underpins multiple cellular processes in A. fumigatus, likely due to the large body of proteins affected by ramA deletion.

A Fungus-Specific Protein Domain Is Essential for RasA-Mediated Morphogenetic Signaling in Aspergillus fumigatus.

Ras proteins function as conserved regulators of eukaryotic growth and differentiation and are essential signaling proteins orchestrating virulence in pathogenic fungi. Here, we report the identification of a novel N-terminal domain of the RasA protein in the filamentous fungus Aspergillus fumigatus. Whereas this domain is absent in Ras homologs of higher eukaryotes, the N-terminal extension is conserved among fungi and is characterized by a short string of two to eight amino acids terminating in an invariant arginine. For this reason, we have termed the RasA N-terminal domain the invariant arginine domain (IRD). Through mutational analyses, the IRD was found to be essential for polarized morphogenesis and asexual development, with the invariant arginine residue being most essential. Although IRD truncation resulted in a nonfunctional Ras phenotype, IRD mutation was not associated with mislocalization of the RasA protein or significant changes in steady-state RasA activity levels. Mutation of the RasA IRD diminished protein kinase A (PKA) activation and resulted in decreased interaction with the Rho-type GTPase, Cdc42. Taken together, our findings reveal novel, fungus-specific mechanisms for Ras protein function and signal transduction. IMPORTANCEAspergillus fumigatus is an important fungal pathogen against which limited treatments exist. During invasive disease, A. fumigatus hyphae grow in a highly polarized fashion, forming filaments that invade blood vessels and disseminate to distant sites. Once invasion and dissemination occur, mortality rates are high. We have previously shown that the Ras signaling pathway is an important regulator of the hyphal growth machinery supporting virulence in A. fumigatus. Here, we show that functional Ras signaling in A. fumigatus requires a novel, fungus-specific domain within the Ras protein. This domain is highly conserved among fungi, yet absent in higher eukaryotes, suggesting a potentially crucial difference in the regulation of Ras pathway activity between the human host and the fungal pathogen. Exploration of the mechanisms through which this domain regulates signaling could lead to novel antifungal therapies specifically targeting fungal Ras pathways.