Monteiro, R. | Search bioRxiv

bioRxiv2018

Genomic analysis of Acinetobacter baumannii prophages reveals remarkable diversity and suggests profound impact on bacterial virulence and fitness

The recent nomination by the World Health Organization of Acinetobacter baumannii as the number one priority pathogen for the development of new antibiotics is a direct consequence of its fast evolution of pathogenicity, and in particular of multidrug resistance. While the development of new antibiotics is critical, understanding the mechanisms behind the crescent bacterial pathogenicity is equally relevant. Often, resistance and other bacterial virulence elements are contained on highly mobile pieces of DNA that can easily spread to other bacteria. Prophages are one of the mediators of this form of gene transfer, and have been frequently found in bacterial genomes, often offering advantageous features to the host. Here we question the contribution of prophages for the evolution of A. baumannii pathogenicity. We found prophages to be notably diverse and widely disseminated in A. baumannii genomes. Also remarkably, A. baumannii prophages encode for multiple putative virulence factors that may be implicated in the bacteriums capacity to colonize host niches, evade the host immune system, subsist in unfavorable environments, and tolerate antibiotics. Overall our results point towards a significant contribution of prophages for the dissemination and evolution of pathogenicity in A. baumannii, and highlight their clinical relevance.

microbiology↗

bioRxiv2017

An optimized pipeline for parallel image-based quantification of gene expression and genotyping after in situ hybridization

Advances in genome engineering have resulted in the generation of numerous zebrafish mutant lines. A commonly used method to assess gene expression in the mutants is in situ hybridization (ISH). Because the embryos can be distinguished by genotype after ISH, comparing gene expression between wild type and mutant siblings can be done blinded and in parallel. Such experimental design reduces the technical variation between samples and minimises the risk of bias. This approach, however, requires an efficient method of genomic DNA extraction from post-ISH fixed zebrafish samples to ascribe phenotype to genotype. Here we describe a method to obtain PCR-quality DNA from 95-100% of zebrafish embryos, suitable for genotyping after ISH. In addition, we provide an image analysis protocol for quantifying gene expression of ISH-probed embryos, adaptable for the analysis of different expression patterns. Finally, we show that intensity-based image analysis enables accurate representation of the variability of gene expression detected by ISH and that it can complement quantitative methods like qRT-PCR. By combining genotyping after ISH and computer-based image analysis, we have established a high-confidence, unbiased methodology to assign gene expression levels to specific genotypes, and applied it to the analysis of molecular phenotypes of newly generated lmo4a mutants.\n\nSUMMARY STATEMENTOur optimized protocol to genotype zebrafish mutant embryos after in situ hybridization and digitally quantify the in situ signal will help to standardize existing experimental designs and methods of analysis.

developmental biology↗