ABSTRACT
Throughout the course of infection, many pathogens encounter bactericidal conditions that threaten the viability of the bacteria and impede the establishment of infection. Bile is one of the most innately bactericidal compounds present in humans, functioning to reduce the bacterial burden in the gastrointestinal tract while also aiding in digestion. It is becoming increasingly apparent that pathogens successfully resist the bactericidal conditions of bile, including bacteria that do not normally cause gastrointestinal infections. This review highlights the ability of Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, Enterobacter (ESKAPE), and other enteric pathogens to resist bile and how these interactions can impact the sensitivity of bacteria to various antimicrobial agents. Given that pathogen exposure to bile is an essential component to gastrointestinal transit that cannot be avoided, understanding how bile resistance mechanisms align with antimicrobial resistance is vital to our ability to develop new, successful therapeutics in an age of widespread and increasing antimicrobial resistance.
Subject(s)
Anti-Bacterial Agents/metabolism , Bacteria/pathogenicity , Bile/metabolism , Drug Resistance, Bacterial , Anti-Bacterial Agents/pharmacology , Bacteria/classification , Bacteria/drug effects , Bacteria/metabolism , Biofilms/drug effects , Biofilms/growth & development , Humans , Intestine, Small/microbiology , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , VirulenceABSTRACT
Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.
Subject(s)
DNA Transposable Elements/genetics , Mutagenesis/genetics , Pseudomonas aeruginosa/chemistry , Animals , Humans , Mutagenesis, InsertionalABSTRACT
Neutrophil breach of the mucosal surface is a common pathological consequence of infection. We present an advanced co-culture model to explore neutrophil transepithelial migration utilizing airway mucosal barriers differentiated from primary human airway basal cells and examined by advanced imaging. Human airway basal cells were differentiated and cultured at air-liquid interface (ALI) on the underside of 3 µm pore-sized transwells, compatible with the study of transmigrating neutrophils. Inverted ALIs exhibit beating cilia and mucus production, consistent with conventional ALIs, as visualized by micro-optical coherence tomography (µOCT). µOCT is a recently developed imaging modality with the capacity for real time two- and three-dimensional analysis of cellular events in marked detail, including neutrophil transmigratory dynamics. Further, the newly devised and imaged primary co-culture model recapitulates key molecular mechanisms that underlie bacteria-induced neutrophil transepithelial migration previously characterized using cell line-based models. Neutrophils respond to imposed chemotactic gradients, and migrate in response to Pseudomonas aeruginosa infection of primary ALI barriers through a hepoxilin A3-directed mechanism. This primary cell-based co-culture system combined with µOCT imaging offers significant opportunity to probe, in great detail, micro-anatomical and mechanistic features of bacteria-induced neutrophil transepithelial migration and other important immunological and physiological processes at the mucosal surface.
