Background: Escherichia coliC forms more robust biofilms than other laboratory strains. Biofilm formation and cell aggregation under a high shear force depend on temperature and salt concentrations. It is the last of fiveE. colistrains (C, K12, B, W, Crooks) designated as safe for laboratory purposes whose genome has not been sequenced.Results: Here we present the complete genomic sequence of this strain in which we utilized both long-read PacBio-based sequencing and high resolution optical mapping to confirm a large inversion in comparison to the other laboratory strains. Notably, DNA sequence comparison revealed the absence of several genes thought to be involved in biofilm formation, including antigen 43,waaSBOJYZULfor lipopolysaccharide (LPS) synthesis, andcpsBfor curli synthesis. The first main difference we identified that likely affects biofilm formation is the presence of an IS3-like insertion sequence in front of the carbon storage regulatorcsrAgene. This insertion is located 86 bp upstream of thecsrAstart codon inside the − 35 region of P4 promoter and blocks the transcription from the sigma³²and sigma⁷⁰promoters P1-P3 located further upstream. The second is the presence of an IS5/IS1182 in front of thecsgDgene. And finally,E. coliC encodes an additional sigma⁷⁰subunit driven by the same IS3-like insertion sequence. Promoter analyses using GFP gene fusions provided insights into understanding this regulatory pathway inE. coli.Conclusions: Biofilms are crucial for bacterial survival, adaptation, and dissemination in natural, industrial, and medical environments. Most laboratory strains ofE. coligrown for decades in vitro have evolved and lost their ability to form biofilm, while environmental isolates that can cause infections and diseases are not safe to work with. Here, we show that the historic laboratory strain ofE. coliC produces a robust biofilm and can be used as a model organism for multicellular bacterial research. Furthermore, we ascertained the full genomic sequence of this classic strain, which provides for a base level of characterization and makes it useful for many biofilm-based applications.

Biofilm infections have no approved effective medical treatments and can only be disrupted via physical means. This means that any biofilm infection that is not addressable surgically can never be eliminated and can only be managed as a chronic disease. Therefore, there is an urgent need for the development of new classes of drugs that can target the metabolic mechanisms within biofilms which render them recalcitrant to traditional antibiotics. Persister cells within the biofilm structure may play a large role in the enhanced antibiotic recalcitrance of bacteria biofilms. Biofilm persister cells can be resistant to up to 1000 times the minimal inhibitory concentrations of many antibiotics, as compared to their planktonic envirovars; they are thought to be the prokaryotic equivalent of metazoan stem cells. Their metabolic resistance has been demonstrated to be an active process induced by the stringent response that is triggered by the ribosomally-associated enzyme RelA in response to amino acid starvation. This 84-kD pyrophosphokinase produces the “magic spot” alarmones, collectively called (p)ppGpp. These alarmones act by directly regulating transcription by binding to RNA polymerase. These transcriptional changes lead to a major shift in cellular function to both upregulate oxidative stress-combating enzymes and down regulate major cellular functions associated with growth and replication. These changes in gene expression produce the quiescent persister cells. In this work, we describe a hybrid in silico laboratory pipeline for identifying and validating small-molecule inhibitors of RelA for use in the combinatorial treatment of bacterial biofilms as re-potentiators of classical antibiotics.

The discovery of profound differences in the brain microbiota of Alzheimer’s disease (AD) patients and age-matched controls (AMCs) raised questions of postmortem contamination and bacterial transport processes which could be informed by microspatial heterogeneities. We performed semiquantitative species-specific bacterial analyses on multiple micro biopsies from each of the 30 brain specimens (AD and controls). We trimmed ~1 mm of each specimen’s edges for surface contaminants and made multiple sterile biopsy punches of the resultant core of each specimen. To identify species-specific abundances, we used our validated, semiquantitative, full-length 16S rRNA gene pan-domain amplification protocol followed by high-fidelity circular consensus sequencing performed on a Pacific Biosciences Sequel IIe instrument. Statistical analyses showed no significant increase in bacterial abundance on trimmed surfaces compared to core specimens, including C. acnes, the most abundant species previously identified in AD. We did find evidence of substantial bacterial species abundance differences among micro-biopsies obtained from within individual tissue blocks supporting our hypothesis of microspatial heterogeneities. The autopsy brain specimens used in our analyses in this study and our previous publication were not contaminated prior to or postharvesting but we suggest that future microbiological analyses of brain specimens include similar types of edge-core comparison analyses. Further, the species-level bacterial abundance heterogeneities among specimens of the same tissue suggest that multiple symbiotic processes may be occurring.