Pseudomonas aeruginosa MPAO1 is the parental strain of the widely utilized transposon mutant collection for this important clinical pathogen. Here, we validate a model system to identify genes involved in biofilm growth and biofilm-associated antibiotic resistance. Our model employs a genomics-driven workflow to assemble the complete MPAO1 genome, identify unique and conserved genes by comparative genomics with the PAO1 reference strain and genes missed within existing assemblies by proteogenomics. Among over 200 unique MPAO1 genes, we identified six general essential genes that were overlooked when mapping public Tn-seq data sets against PAO1, including an antitoxin. Genomic data were integrated with phenotypic data from an experimental workflow using a user-friendly, soft lithography-based microfluidic flow chamber for biofilm growth and a screen with the Tn-mutant library in microtiter plates. The screen identified hitherto unknown genes involved in biofilm growth and antibiotic resistance. Experiments conducted with the flow chamber across three laboratories delivered reproducible data on P. aeruginosa biofilms and validated the function of both known genes and genes identified in the Tn-mutant screens. Differential Protein abundance data from planktonic cells versus biofilm confirmed the upregulation of candidates known to affect biofilm formation, of structural and secreted proteins of type VI secretion systems, and provided proteogenomic evidence for some missed MPAO1 genes. This integrated, broadly applicable model promises to improve the mechanistic understanding of biofilm formation, antimicrobial tolerance, and resistance evolution in biofilms.
Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance.
This report is a literature review of the current state of knowledge of fireworks
and fireworks hazards. It presents work to meet Deliverables D4-1 (types and compositions
of fireworks Articles), D4-2 (Functioning mechanism of and between fireworks
articles), D4-3 (Storage and transport regulations in EU countries) and D4-4 (The effect
of fireworks on health and the environment) of the CHAF work program.
Firework compositions are discussed in the first section. This leads to an understanding
of the variability in performance of various types of fireworks. Compositions
used in report effects are highlighted as being the most energetic while other compositions
are seen as providing a much lesser hazard in mass storage of fireworks. Individual
types of firework are examined. Large devices such as report shells are identified as
posing the largest hazard in bulk storage. Lesser, but still significant, hazards are predicted
for similar compositions in Roman candles and rockets. Gradations in hazard are
identified corresponding to UN hazard divisions 1.1, 1.3 and 1.4.
The second section reports the literature relating to the functioning mechanism of
and between fireworks articles. In this the major propagation mechanisms for fireworks
compositions are examined. The main stimuli identified are: fragment impact, heat and
shock wave, each of which can lead to the functioning of pyrotechnic composition in
the article or in an adjacent article leading to large-scale initiation. Consideration of the
pyrotechnic compositions leads to a different ranking for the three mechanisms with different
fireworks types. Thus with a report composition containing a metal/perchlorate
mixture shock initiation is seen to be the main propagation mechanism. Such a mechanism
is likely to afford a mass explosion. At a lesser hazard, blackpowder-based compositions
are reported to be more likely to propagate via a flame mechanism. This is less
likely to produce a mass explosion.
National legislation concerning the storage of fireworks for many European countries
is considered in the third section. Many of the national regulations are based on a
cube root of explosives content to determine "safety distances", often written as Q1/3.
Different multiplying factors are then applied depending on the type of adjacent site
(production buildings, residential housing, roads etc.). Commonly 22Q1/3 is used for residential
dwellings. Special provisions are enacted in many countries either to allow
lesser distances or in some cases to increase the safety distances depending on the
perceived hazard or extenuating circumstances.
The final section deals with the environmental hazards posed by the use of
fireworks. Toxic effects of the materials used in the manufacture and the likely reaction
products from functioning fireworks are highlighted. These range from heavy metal
poisoning from metals present for their colour effect to respiratory problems due to
pyrotechnic fume (both particulate and gaseous). Finally, noise pollution is considered.
While there is a short term problem from pyrotechnic fume at times of high usage
(national celebrations) there is no literature evidence, as yet, of long term detriment to
the environment.
A systematic study of annealing behavior of drawn PMMA fibers was performed. Annealing Dynamics were investigated under different environmental conditions by fiber longitudinal shrinkage monitoring. The shrinkage process was found to follow a stretched exponential decay function revealing the heterogeneous nature of the underlying molecular dynamics. The complex dependence of the fiber shrinkage on initial degree of molecular alignment in the fiber, annealing time and temperature was investigated and interpreted. Moreover, humidity was shown to have a profound effect on the annealing process, which was not recognized previously. Annealing was also shown to have considerable effect on the fiber mechanical properties associated with the relaxation of molecular alignment in the fiber. The consequences of fiber annealing for the climatic stability of certain polymer optical fiber-based sensors are discussed, emphasizing the importance of fiber controlled pre-annealing with respect to the foreseeable
operating conditions.