The Contribution of Mobile Genetic Elements to the Molecular Biology of Streptococcus pyogenes Genotype emm4

Genotype emm4 Streptococcus pyogenes constitute a major disease-causing emm-type worldwide, and have been associated with scarlet fever. Genotype emm4 are lysogenised by three toxigenic prophage, and an atoxigenic SpyCI, thought to regulate the DNA mismatch repair operon by growth-phase dependent excision. Phylogenetic analyses determined that emm4 in our collection and internationally, clustered with either reference genomes MGAS10750 or MEW427, the latter of which were found to have undergone substantial gene loss within prophage-encoding regions. Gene loss affected modules pertaining to lysogeny, genetic regulation, replication, and encapsidation, but not cognate toxins. Corresponding elements of MGAS10750-like emm4 were full-length and undegraded. The lineages associated with either MGAS10750 or MEW427 were thus denoted M4complete and M4degraded, respectively. Prophage of M4degraded isolates were shown to be cryptic, and incapable of excision, whereas prophage of M4complete isolates were inducible. Unexpectedly, this had little effect on cognate toxin expression, which was independent of induction and replication. Further, the emm4 SpyCI was found to be immobile, irrespective of gene content, and appears not to regulate the DNA MMR operon, as had been reported. A number of assays did not demonstrate lineage-specific phenotypes, however, M4complete isolates were significantly more virulent than M4degraded isolates in a Galleria mellonella infection model. Intriguingly, efforts to explore this response in more infection-relevant conditions revealed that co-culture with human tonsil cells greatly enhanced expression of the prophage-associated superantigen ssa, among both M4complete and M4degraded isolates, and this was also observed among emm3 and emm12. There may also be a hitherto unappreciated role for major bacterial regulators in the control and elicitation of this response. These data suggest that the relationship between S. pyogenes, their viral parasites and the human host may be much more complex than had been anticipated, and present a number of exciting avenues for further study

The DNases of pathogenic Lancefield streptococci

DNases are abundant among the pathogenic streptococci, with most species harbouring genes for at least one. Despite their prevalence, however, the role for these extracellular enzymes is still relatively unclear. The DNases of the Lancefield group A Streptococcus, S. pyogenes are the best characterized, with a total of eight DNase genes identified so far. Six are known to be associated with integrated prophages. Two are chromosomally encoded, and one of these is cell-wall anchored. Homologues of both prophage-associated and chromosomally encoded S. pyogenes DNases have been identified in other streptococcal species, as well as other unique DNases. A major role identified for streptococcal DNases appears to be in the destruction of extracellular traps produced by immune cells, such as neutrophils, to ensnare bacteria and kill them. These traps are composed primarily of DNA which can be degraded by the secreted and cell-wall-anchored streptococcal DNases. DNases can also reduce TLR-9 signalling to dampen the immune response and produce cytotoxic deoxyadenosine to limit phagocytosis. Upper respiratory tract infection models of S. pyogenes have identified a role for DNases in potentiating infection and transmission, possibly by limiting the immune response or through some other unknown mechanism. Streptococcal DNases may also be involved in interacting with other microbial communities through communication, bacterial killing and disruption of competitive biofilms, or control of their own biofilm production. The contribution of DNases to pathogenesis may therefore be wide ranging and extend beyond direct interference with the host immune response

Correlation of Parameters used to Estimate Monomer Conversion in a Light-cured Composite

The sensitivities of Fourier transform infrared spectroscopy, Knoop hardness, water sorption, and resin leaching were compared for their ability to distinguish differences between composite samples cured through different thicknesses of overlying resin. The method developed allowed samples of light-cured composite to be made with controlled conversion for parameter testing, and eliminated effects of resin lost to slurry during polishing or an increase in conversion as a result of heat generated during grinding. Sensitivity to differences was greatest and equal for FTIR spectroscopy and Knoop hardness, while resin leaching proved to have moderate sensitivity, and water sorption none. The ability of these parameters to predict monomer conversion as measured by FTIR spectroscopy was also determined. Knoop hardness proved the best conversion predictor, resin leaching the next best, and water sorption the worst. Water sorption values did not vary with changes in specimen conversion.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/67029/2/10.1177_00220345880670060801.pd

Cryptic prophages within a Streptococcus pyogenes genotype emm4 lineage

The major human pathogen Streptococcus pyogenes shares an intimate evolutionary history with mobile genetic elements, which in many cases carry genes encoding bacterial virulence factors. During recent whole-genome sequencing of a longitudinal sample of S. pyogenes isolates in England, we identified a lineage within emm4 that clustered with the reference genome MEW427. Like MEW427, this lineage was characterized by substantial gene loss within all three prophage regions, compared to MGAS10750 and isolates outside of the MEW427-like lineage. Gene loss primarily affected lysogeny, replicative and regulatory modules, and to a lesser and more variable extent, structural genes. Importantly, prophage-encoded superantigen and DNase genes were retained in all isolates. In isolates where the prophage elements were complete, like MGAS10750, they could be induced experimentally, but not in MEW427-like isolates with degraded prophages. We also found gene loss within the chromosomal island SpyCIM4 of MEW427-like isolates, although surprisingly, the SpyCIM4 element could not be experimentally induced in either MGAS10750-like or MEW427-like isolates. This did not, however, appear to abolish expression of the mismatch repair operon, within which this element resides. The inclusion of further emm4 genomes in our analyses ratified our observations and revealed an international emm4 lineage characterized by prophage degradation. Intriguingly, the USA population of emm4 S. pyogenes appeared to constitute predominantly MEW427-like isolates, whereas the UK population comprised both MEW427-like and MGAS10750-like isolates. The degraded and cryptic nature of these elements may have important phenotypic and fitness ramifications for emm4 S. pyogenes , and the geographical distribution of this lineage raises interesting questions on the population dynamics of the genotype

Targeting biofilms using phages and their enzymes

"Available online 10 March 2021"The complex biofilm architecture composed of extracellular polymeric structures (EPS) provides a protective shield to physiologically diverse bacterial cells immersed in its structure. The evolutionary interplay between bacteria and their viruses (phages) forced the latter ones to develop specific strategies to overcome the biofilm defensive barriers and kill sessile cells. Phages are equipped with a wide panel of enzyme-degrading EPS macromolecules which together are powerful weapons to combat biofilms. Antibiofilm performance can be achieved by combining phages or phage-borne enzymes with other antimicrobials such as antibiotics. Nevertheless, a variety of enzymes encoded in phage genomes still need to be explored. To advance in biofilm control strategies we must deepen the understanding of the biofilm biology itself, as well as discover and better exploit the unlimited antibacterial potential of phages.JA was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit. PG was supported by grants from Innovative Training Networks (ITN) Marie Skłodowska-Curie Actions H2020-MSCA-ITN2018. Reference 813439, and PID2019-105311RB-I00 (MICIU/AEI/FEDER, UE, Spain). ZDK was supported by the National Science Centre of Poland (Narodowe Centrum Nauki), grant numbers 2016/21/B/NZ6/01157 and 2017/26/M/NZ1/00233.info:eu-repo/semantics/publishedVersio

Activity and Interactions of Liposomal Antibiotics in Presence of Polyanions and Sputum of Patients with Cystic Fibrosis

BACKGROUND:To compare the effectiveness of liposomal tobramycin or polymyxin B against Pseudomonas aeruginosa in the Cystic Fibrosis (CF) sputum and its inhibition by common polyanionic components such as DNA, F-actin, lipopolysaccharides (LPS), and lipoteichoic acid (LTA). METHODOLOGY:Liposomal formulations were prepared from a mixture of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) or 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) and Cholesterol (Chol), respectively. Stability of the formulations in different biological milieus and antibacterial activities compared to conventional forms in the presence of the aforementioned inhibitory factors or CF sputum were evaluated. RESULTS:The formulations were stable in all conditions tested with no significant differences compared to the controls. Inhibition of antibiotic formulations by DNA/F-actin and LPS/LTA was concentration dependent. DNA/F-actin (125 to 1000 mg/L) and LPS/LTA (1 to 1000 mg/L) inhibited conventional tobramycin bioactivity, whereas, liposome-entrapped tobramycin was inhibited at higher concentrations--DNA/F-actin (500 to 1000 mg/L) and LPS/LTA (100 to 1000 mg/L). Neither polymyxin B formulation was inactivated by DNA/F-actin, but LPS/LTA (1 to 1000 mg/L) inhibited the drug in conventional form completely and higher concentrations of the inhibitors (100 to 1000 mg/L) was required to inhibit the liposome-entrapped polymyxin B. Co-incubation with inhibitory factors (1000 mg/L) increased conventional (16-fold) and liposomal (4-fold) tobramycin minimum bactericidal concentrations (MBCs), while both polymyxin B formulations were inhibited 64-fold. CONCLUSIONS:Liposome-entrapment reduced antibiotic inhibition up to 100-fold and the CFU of endogenous P. aeruginosa in sputum by 4-fold compared to the conventional antibiotic, suggesting their potential applications in CF lung infections

Evidence for perinatal and child health care guidelines in crisis settings: can Cochrane help?

<p>Abstract</p> <p>Background</p> <p>It is important that healthcare provided in crisis settings is based on the best available research evidence. We reviewed guidelines for child and perinatal health care in crisis situations to determine whether they were based on research evidence, whether Cochrane systematic reviews were available in the clinical areas addressed by these guidelines and whether summaries of these reviews were provided in Evidence Aid.</p> <p>Methods</p> <p>Broad internet searches were undertaken to identify relevant guidelines. Guidelines were appraised using AGREE and the clinical areas that were relevant to perinatal or child health were extracted. We searched The Cochrane Database of Systematic Reviews to identify potentially relevant reviews. For each review we determined how many trials were included, and how many were conducted in resource-limited settings.</p> <p>Results</p> <p>Six guidelines met selection criteria. None of the included guidelines were clearly based on research evidence. 198 Cochrane reviews were potentially relevant to the guidelines. These reviews predominantly addressed nutrient supplementation, breastfeeding, malaria, maternal hypertension, premature labour and prevention of HIV transmission. Most reviews included studies from developing settings. However for large portions of the guidelines, particularly health services delivery, there were no relevant reviews. Only 18 (9.1%) reviews have summaries in Evidence Aid.</p> <p>Conclusions</p> <p>We did not identify any evidence-based guidelines for perinatal and child health care in disaster settings. We found many Cochrane reviews that could contribute to the evidence-base supporting future guidelines. However there are important issues to be addressed in terms of the relevance of the available reviews and increasing the number of reviews addressing health care delivery.</p

Erratum to: 36th International Symposium on Intensive Care and Emergency Medicine

[This corrects the article DOI: 10.1186/s13054-016-1208-6.]

Complementing a Forage Program with Annual Forages

Economists tell us that in a mature industry you must be a low cost producer to survive. The meat industry is ending the century much more mature than it began. Sophisticated production of pork and poultry with the benefit of cheap grain is putting pressure on beef producers. The long-term trend in grain prices is downward because the cost of producing grain is being reduced by three ongoing trends. The continuing enrichment of the atmosphere by CO2 and other greenhouse gases is crop production friendly despite what you may have heard of few years back. Secondly, bio-tech seeds are increasing yields and decreasing unit cost of production. Additionally, governments here and abroad are continuing to subsidize grain producers. This suggests to me that we, as forage based calf producers, must increase our sophistication and efficiency in feeding the brood cow. Forages are only cheap in relationship to grain when they are directly harvested by the cow. We have come a long ways in the last few decades in reducing the amount of put up hay we feed our cows. To compete with grain based meat production in the new century and millennium we must continue to improve our nutritional management. Just because you can put up and feed back hundreds of tons of forage, and hardly leave an air-conditioned tractor seat, doesn\u27t mean you can afford it. You may need to sell most of your high quality hay to the dairy or horse industry down the road and look for corn stalks and other cheaper ways of feeding your cows. I want to share with you how I use a small amount of crop acreage to enhance the economic benefits of a forage harvesting cow herd

LO-CO-GRAF: generating maps to support command & control/crisis management using small computers

This thesis describes and tests a system for Low Cost Graphics (LO- CO-GRAF) which enables a small computer to generate and display high quality cartographic maps from a remote mainframe computer database. A small computer must emulate a graphics terminal while interfacing with a mainframe program which processes the necessary data. This solution has been accomplished through four smaller tasks. The tasks include communicating with the source system, emulating a graphics terminal, interfacing with a map generation program, and producing a local hardcopy of the generated map. All software and hardware required for these component parts, in addition to the use of standard methodologies, were selected for their widespread availability at Department of Defense (DOD) agencies. Research was conducted using the Health/Zenith Z-120 as the small microcomputer and the Tektronix 4010 graphics terminal was chosen to model and emulate; two separate source graphics packages were used to generate maps. Concept validation involved the use of DISSPLA, the primary graphics package used on an IBM 3033 mainframe computer at the Naval Postgraduate School, and the Briefing Aid System, a map generation program maintained on the VAX mainframe computer at University of Southern California's Information Systems Institute, which was accessed and employed via Defense Data Network (DDN)http://archive.org/details/locografgenerati00saboU.S. Navy (USN) autho