Cell wall deficiency – an alternate bacterial lifestyle?

Historically, many species of bacteria have been reported to produce viable, cell wall deficient (CWD) variants. A variety of terms have been used to refer to CWD bacteria and a plethora of methods described in which to induce, cultivate and propagate them. In this review, we will examine the long history of scientific research on CWD bacteria examining the methods by which CWD bacteria are generated; the requirements for survival in a CWD state; the replicative processes within a CWD state; and the reversion of CWD bacteria into a walled state, or lack thereof. In doing so, we will present evidence that not all CWD variants are alike and that, at least in some cases, CWD variants arise through an adaptive lifestyle switch that enables them to live and thrive without a cell wall, often to avoid antimicrobial activity. Finally, the implications of CWD bacteria in recurring infections, tolerance to antibiotic therapy and antimicrobial resistance will be examined to illustrate the importance of greater understanding of the CWD bacteria in human health and disease

Pseudomonas aeruginosa is capable of natural transformation in biofilms

Natural transformation is a mechanism that enables competent bacteria to acquire naked, exogenous DNA from the environment. It is a key process that facilitates the dissemination of antibiotic resistance and virulence determinants throughout bacterial populations. Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that produces large quantities of extracellular DNA (eDNA) that is required for biofilm formation. P. aeruginosa has a remarkable level of genome plasticity and diversity that suggests a high degree of horizontal gene transfer and recombination but is thought to be incapable of natural transformation. Here we show that P. aeruginosa possesses homologues of all proteins known to be involved in natural transformation in other bacterial species. We found that P. aeruginosa in biofilms is competent for natural transformation of both genomic and plasmid DNA. Furthermore, we demonstrate that type-IV pili (T4P) facilitate but are not absolutely essential for natural transformation in P. aeruginosa

A computable pathology report for precision medicine: extending an observables ontology unifying SNOMED CT and LOINC

Background The College of American Pathologists (CAP) introduced the first cancer synoptic reporting protocols in 1998. However, the objective of a fully computable and machine-readable cancer synoptic report remains elusive due to insufficient definitional content in Systematized Nomenclature of Medicine – Clinical Terms (SNOMED CT) and Logical Observation Identifiers Names and Codes (LOINC). To address this terminology gap, investigators at the University of Nebraska Medical Center (UNMC) are developing, authoring, and testing a SNOMED CT observable ontology to represent the data elements identified by the synoptic worksheets of CAP. Methods Investigators along with collaborators from the US National Library of Medicine, CAP, the International Health Terminology Standards Development Organization, and the UK Health and Social Care Information Centre analyzed and assessed required data elements for colorectal cancer and invasive breast cancer synoptic reporting. SNOMED CT concept expressions were developed at UNMC in the Nebraska Lexicon© SNOMED CT namespace. LOINC codes for each SNOMED CT expression were issued by the Regenstrief Institute. SNOMED CT concepts represented observation answer value sets. Results UNMC investigators created a total of 194 SNOMED CT observable entity concept definitions to represent required data elements for CAP colorectal and breast cancer synoptic worksheets, including biomarkers. Concepts were bound to colorectal and invasive breast cancer reports in the UNMC pathology system and successfully used to populate a UNMC biobank. Discussion The absence of a robust observables ontology represents a barrier to data capture and reuse in clinical areas founded upon observational information. Terminology developed in this project establishes the model to characterize pathology data for information exchange, public health, and research analytics

Multiple holins contribute to extracellular DNA release in Pseudomonas aeruginosa biofilms

Bacterial biofilms are composed of aggregates of cells encased within a matrix of extracellular polymeric substances (EPS). One key EPS component is extracellular DNA (eDNA), which acts as a ‘glue’, facilitating cell–cell and cell–substratum interactions. We have previously demonstrated that eDNA is produced in Pseudomonas aeruginosa biofilms via explosive cell lysis. This phenomenon involves a subset of the bacterial population explosively lysing, due to peptidoglycan degradation by the endolysin Lys. Here we demonstrate that in P. aeruginosa three holins, AlpB, CidA and Hol, are involved in Lys-mediated eDNA release within both submerged (hydrated) and interstitial (actively expanding) biofilms, albeit to different extents, depending upon the type of biofilm and the stage of biofilm development. We also demonstrate that eDNA release events determine the sites at which cells begin to cluster to initiate microcolony formation during the early stages of submerged biofilm development. Furthermore, our results show that sustained release of eDNA is required for cell cluster consolidation and subsequent microcolony development in submerged biofilms. Overall, this study adds to our understanding of how eDNA release is controlled temporally and spatially within P. aeruginosa biofilms

A feedheat-integrated energy storage system for nuclear-powered steam plant

Peer reviewed: TrueThe paper provides thermodynamic analysis of an energy storage concept in which thermal stores are coupled with the feedwater heating train of nuclear-powered steam plant. This allows the electrical output of the plant to be flexed whilst maintaining constant reactor power, thereby providing the equivalent of an electricity storage system and facilitating the adoption of a load-following role for nuclear plant. The concept falls under the umbrella of ‘generation-integrated energy storage’, which exploits existing hardware required for generation to reduce storage costs, and benefits also from fewer energy transformations when compared to ‘electricity-in-electricity-out’ forms of storage. This means that a high (effective) round-trip efficiency can be achieved at low cost. An important feature of the proposed system is that the turbine bleed flows (at their various pressures and temperatures) automatically provide good thermal matching with the feedwater temperature profile, so that heat can ultimately be transferred to and from sensible-heat thermal-storage media with high exergetic efficiency. Various options are discussed for the thermal stores, including pressurised water tanks, thermal oils and packed beds. The analysis also includes consideration of the off-design behaviour of cycle components

A quadruple knockout of LasIR and rhlIR of Pseudomonas aeruginosa PAO1 that retains wild-type twitching motility has equivalent infectivity and persistence to PAO1 in a mouse model of lung infection

It has been widely reported that quorum-sensing incapable strains of Pseudomonas aeruginosa are less virulent than wildtype strains. However, quorum sensing mutants of P. aeruginosa have been shown to develop other spontaneous mutations under prolonged culture conditions, and one of the phenotypes of P. aeruginosa that is frequently affected by this phenomenon is type IV pili-dependent motility, referred to as twitching motility. As twitching motility has been reported to be important for adhesion and colonisation, we aimed to generate a quorum-sensing knockout for which the heritage was recorded and the virulence factor production in areas unrelated to quorum sensing was known to be intact. We created alas IRrhlIR quadruple knockout in PAO1 using a published technique that allows for the deletion of antibiotic resistancecartridges following mutagenesis, to create an unmarked QS knockout of PAO1, thereby avoiding the need for use ofantibiotics in culturing, which can have subtle effects on bacterial phenotype. We phenotyped this mutant demonstratingthat it produced reduced levels of protease and elastase, barely detectable levels of pyoverdin and undetectable levels ofthe quorum sensing signal molecules N-3-oxododecanoly-L-homoserine lactone and N-butyryl homoserine lactone, butretained full twitching motility. We then used a mouse model of acute lung infection with P. aeruginosa to demonstrate that the lasIRrhlIR knockout strain showed equal persistence to wild type parental PAO1, induced equal or greater neutrophilin filtration to the lungs, and induced similar levels of expression of inflammatory cytokines in the lungs and similar antibody responses, both in terms of magnitude and isotype. Our results suggest, in contrast to previous reports, that lack of quorum sensing alone does not significantly affect the immunogenicity, infectiveness and persistence of P. aeruginosa in a mouse model of acute lung infection

Anti-<i>Pseudomonas</i> antibody production by mice infected with PAO1 or PAO1Δ<i>lasIRrhlIR</i>.

<p>Mice were infected intratracheally with 1×10³ CFU of PAO1 or PAO1Δ<i>lasIRrhlIR</i> in an 0.5% agar slurry, and bled on day 22. Sera were tested in ELISA against antigen prepared from whole killed PAO1, using isotype-specific detection antibodies for mouse IgM, IgG1, and IgG2a. Results are expressed as individual titers with a line representing the median values. There were no significant differences (Mann–Whitney <i>U</i> test) between mice infected with PAO1 or PAO1Δ<i>lasIRrhlIR</i> (QSneg).</p

Expression of cytokine mRNA in lung tissue of mice 4 h after infection with PAO1 or PAO1Δ<i>lasIRrhlIR</i>.

<p>Lung tissue from mice infected with 1×10⁶ CFU of either PAO1 (hatched bars) or PAO1Δ<i>lasIRrhlI</i> (QSneg, open bars) was processed, total RNA extracted and cDNA prepared. Expression of cytokine genes was assessed in RT-qPCR using β-actin as the reference gene. All results are expressed as gene expression relative to β-actin, and are presented as the mean ± SD of five mice per group. A control group of lungs from mice that underwent anaesthesia and intratracheal infusion of PBS without bacteria was included (grey bars). Expression of all cytokines was significantly higher in mice infected with bacteria than in sham-infected mice (<i>P</i><0.01, all groups, ANOVA) but there were no significant differences between mice infected with PAO1 or PAO1Δ<i>lasIRrhlIR</i> (QSneg).</p

Primers used in this study.

<p>Primers used in this study.</p

Production of C4HSL and 3OC12HSL by QS mutants compared with PAO1.

<p>The capacity of all mutants to produce C4HSL (a) and 3OC12HSL (b) is presented. Because of interassay variability, all results are presented as percent of production by wild type PAO1. Results for 100 µM C4HSL (a) or 3OC12HSL (b) included as positive controls. Values represent mean ± SD of at least triplicate determinations. LB: LB broth (negative control). ND: not detected.</p