High-precision screening and sorting of double emulsion droplets.

Mounting evidence suggests that cell populations are extremely heterogeneous, with individual cells fulfilling different roles within the population. Flow cytometry (FC) is a high-throughput tool for single-cell analysis that works at high optical resolution. Sub-populations with unique properties can be screened, isolated and sorted through fluorescence-activated cell sorting (FACS), using intracellular fluorescent products or surface-tagged fluorescent products of interest. However, traditional FC and FACS methods cannot identify or isolate cells that secrete extracellular products of interest. Double emulsion (DE) droplets are an innovative approach to retaining these extracellular products so cells producing them can be identified and isolated with FC and FACS. The water-in-oil-in-water structure makes DE droplets compatible with the sheath flow of flow cytometry. Single cells can be encapsulated with other reagents into DEs, which act as pico-reactors. These droplets allow biological activities to take place while allowing for cell cultivation monitoring, rare mutant identification, and cellular events characterization. However, using DEs in FACS presents technical challenges, including rupture of DEs, poor accuracy and low sorting efficiency. This study presents high-performance sorting using fluorescent beads (as simulants for cells). This study aims to guide researchers in the use of DE-based flow cytometry, offering insights into how to resolve the technical difficulties associated with DE-based screening and sorting using FC.

Systematic analyses identify modes of action of ten clinically relevant biocides and antibiotic antagonism in Acinetobacter baumannii.

Concerns exist that widespread use of antiseptic or disinfectant biocides could contribute to the emergence and spread of multidrug-resistant bacteria. To investigate this, we performed transposon-directed insertion-site sequencing (TraDIS) on the multidrug-resistant pathogen, Acinetobacter baumannii, exposed to a panel of ten structurally diverse and clinically relevant biocides. Multiple gene targets encoding cell envelope or cytoplasmic proteins involved in processes including fatty acid biogenesis, multidrug efflux, the tricarboxylic acid cycle, cell respiration and cell division, were identified to have effects on bacterial fitness upon biocide exposure, suggesting that these compounds may have intracellular targets in addition to their known effects on the cell envelope. As cell respiration genes are required for A. baumannii fitness in biocides, we confirmed that sub-inhibitory concentrations of the biocides that dissipate membrane potential can promote A. baumannii tolerance to antibiotics that act intracellularly. Our results support the concern that residual biocides might promote antibiotic resistance in pathogenic bacteria.

Rapid microevolution of biofilm cells in response to antibiotics.

Infections caused by Acinetobacter baumannii are increasingly antibiotic resistant, generating a significant public health problem. Like many bacteria, A. baumannii adopts a biofilm lifestyle that enhances its antibiotic resistance and environmental resilience. Biofilms represent the predominant mode of microbial life, but research into antibiotic resistance has mainly focused on planktonic cells. We investigated the dynamics of A. baumannii biofilms in the presence of antibiotics. A 3-day exposure of A. baumannii biofilms to sub-inhibitory concentrations of antibiotics had a profound effect, increasing biofilm formation and antibiotic resistance in the majority of biofilm dispersal isolates. Cells dispersing from biofilms were genome sequenced to identify mutations accumulating in their genomes, and network analysis linked these mutations to their phenotypes. Transcriptomics of biofilms confirmed the network analysis results, revealing novel gene functions of relevance to both resistance and biofilm formation. This approach is a rapid and objective tool for investigating resistance dynamics of biofilms.