New proof-of-concept in viral inactivation: virucidal efficacy of 405 nm light against feline calicivirus as a model for norovirus decontamination

The requirement for novel decontamination technologies for use in hospitals is ever present. One such system uses 405 nm visible light to inactivate microorganisms via ROS-generated oxidative damage. Although effective for bacterial and fungal inactivation, little is known about the virucidal effects of 405 nm light. Norovirus (NoV) gastroenteritis outbreaks often occur in the clinical setting, and this study was designed to investigate potential inactivation effects of 405 nm light on the NoV surrogate, feline calicivirus (FCV). FCV was exposed to 405 nm light whilst suspended in minimal and organically-rich media to establish the virucidal efficacy and the effect biologically-relevant material may play in viral susceptibility. Antiviral activity was successfully demonstrated with a 4 Log10 (99.99%) reduction in infectivity when suspended in minimal media evident after a dose of 2.8 kJ cm−2. FCV exposed in artificial faeces, artificial saliva, blood plasma and other organically rich media exhibited an equivalent level of inactivation using between 50–85% less dose of the light, indicating enhanced inactivation when the virus is present in organically-rich biologically-relevant media. Further research in this area could aid in the development of 405 nm light technology for effective NoV decontamination within the hospital environment

Decontamination of the hospital environment : new technologies for infection control

Environmental contamination is being increasingly recognized as a significant source of healthcare-associated infection (HAI). Cross-contamination of the patient from the environment can result from the direct transfer of organisms from the air and surfaces, or indirectly from the hospital environment via contact with healthcare workers or equipment. Traditional methods of environmental decontamination, including cleaning with disinfectants, and the standard infection control procedures implemented by modern Health Services, are critical to controlling the spread of potentially pathogenic microbial contaminants from environmental sources to the patient; however there is constant pressure to maintain, and indeed, improve on the standards that are in place to ensure optimal patient care. To address this issue, much research has been directed towards the development and testing of novel ‘whole-room’ environmental decontamination methods which could be used to enhance hospital hygiene, and consequently reduce the risk of HAI-acquisition from environmental sources. Gaseous methods such as the use of hydrogen peroxide, chlorine dioxide, ozone and steam, as well as ultraviolet and violet-blue visible light methods have all been laboratory tested, and to varying extents, clinically evaluated to assess their efficacy for environmental decontamination. This review article considers these different decontamination technologies, discussing their mechanism of action, antimicrobial efficacy, and advantages and limitations, with a view to providing the reader with a comprehensive overview of the technological advances being developed to reduce the levels of environmental contamination around patient areas, thus aiding in the fight against healthcare-associated infection

Inactivation of Streptomyces phage ɸC31 by 405 nm light : requirement for exogenous photosensitizers?

Exposure to narrowband violet-blue light around 405 nm wavelength can induce lethal oxidative damage to bacteria and fungi, however effects on viruses are unknown. As photosensitive porphyrin molecules are involved in the microbicidal inactivation mechanism, and since porphyrins are absent in viruses, then any damaging effects of 405 nm light on viruses might appear unlikely. This study used the bacteriophage ɸC31, as a surrogate for non-enveloped double-stranded DNA viruses, to establish whether 405 nm light can induce virucidal effects. Exposure of ɸC31 suspended in minimal media, nutrient-rich media, and porphyrin solution, demonstrated differing sensitivity of the phage. Significant reductions in phage titre occurred when exposed in nutrient-rich media, with ~3, 5 and 7-log10 reductions achieved after exposure to doses of 0.3, 0.5 and 1.4 kJ/cm2, respectively. When suspended in minimal media a 0.3 log10 reduction (P=0.012) occurred after exposure to 306 J/cm2: much lower than the 2.7 and >2.5 log10 reductions achieved with the same dose in nutrient-rich, and porphyrin-supplemented media, suggesting inactivation is accelerated by the photo-activation of light-sensitive components in the media. This study provides the first evidence of the interaction of narrowband 405 nm light with viruses, and demonstrates that viral susceptibility to 405 nm light can be significantly enhanced by involvement of exogenous photosensitive components. The reduced susceptibility of viruses in minimal media, compared to that of other microorganisms, provides further evidence that the antimicrobial action of 405 nm light is predominantly due to the photo-excitation of endogenous photosensitive molecules such as porphyrins within susceptible microorganisms

Non-ionizing 405nm light as a potential bactericidal technology for platelet safety : evaluation of in vitro bacterial inactivation and in vivo platelet recovery in severe combined immunodeficient mice

Bacterial contamination of ex vivo stored platelets is a cause of transfusion-transmitted infection. Violet-blue 405 nm light has recently demonstrated efficacy in reducing the bacterial burden in blood plasma, and its operational benefits such as non-ionizing nature, penetrability, and non-requirement for photosensitizing agents, provide a unique opportunity to develop this treatment for in situ treatment of ex vivo stored platelets as a tool for bacterial reduction. Sealed bags of platelet concentrates, seeded with low-level Staphylococcus aureus contamination, were 405 nm light-treated (3-10 mWcm-2) up to 8 hr. Antimicrobial efficacy and dose efficiency was evaluated by quantification of the post-treatment surviving bacterial contamination levels. Platelets treated with 10 mWcm-2 for 8 hr were further evaluated for survival and recovery in severe combined immunodeficient (SCID) mice. Significant inactivation of bacteria in platelet concentrates was achieved using all irradiance levels, with 99.6-100% inactivation achieved by 8 hr (P<0.05). Analysis of applied dose demonstrated that lower irradiance levels generally resulted in significant decontamination at lower doses: 180 Jcm-2/10 mWcm-2 (P=0.008) compared to 43.2 Jcm-2/3 mWcm-2 (P=0.002). Additionally, the recovery of light-treated platelets, compared to non-treated platelets, in the murine model showed no significant differences (P ≥ 0.05). This report paves the way for further comprehensive studies to test 405 nm light treatment as a bactericidal technology for stored platelet

Mosaic DNA imports with interspersions of recipient sequence after natural transformation of Helicobacter pylori

Helicobacter pylori colonizes the gastric mucosa of half of the human population, causing gastritis, ulcers, and cancer. H. pylori is naturally competent for transformation by exogenous DNA, and recombination during mixed infections of one stomach with multiple H. pylori strains generates extensive allelic diversity. We developed an in vitro transformation protocol to study genomic imports after natural transformation of H. pylori. The mean length of imported fragments was dependent on the combination of donor and recipient strain and varied between 1294 bp and 3853 bp. In about 10% of recombinant clones, the imported fragments of donor DNA were interrupted by short interspersed sequences of the recipient (ISR) with a mean length of 82 bp. 18 candidate genes were inactivated in order to identify genes involved in the control of import length and generation of ISR. Inactivation of the antimutator glycosylase MutY increased the length of imports, but did not have a significant effect on ISR frequency. Overexpression of mutY strongly increased the frequency of ISR, indicating that MutY, while not indispensable for ISR formation, is part of at least one ISR-generating pathway. The formation of ISR in H. pylori increases allelic diversity, and contributes to the uniquely low linkage disequilibrium characteristic of this pathogen

Microevolution of Helicobacter pylori during prolonged infection of single hosts and within families

Our understanding of basic evolutionary processes in bacteria is still very limited. For example, multiple recent dating estimates are based on a universal inter-species molecular clock rate, but that rate was calibrated using estimates of geological dates that are no longer accepted. We therefore estimated the short-term rates of mutation and recombination in Helicobacter pylori by sequencing an average of 39,300 bp in 78 gene fragments from 97 isolates. These isolates included 34 pairs of sequential samples, which were sampled at intervals of 0.25 to 10.2 years. They also included single isolates from 29 individuals (average age: 45 years) from 10 families. The accumulation of sequence diversity increased with time of separation in a clock-like manner in the sequential isolates. We used Approximate Bayesian Computation to estimate the rates of mutation, recombination, mean length of recombination tracts, and average diversity in those tracts. The estimates indicate that the short-term mutation rate is 1.4×10−6 (serial isolates) to 4.5×10−6 (family isolates) per nucleotide per year and that three times as many substitutions are introduced by recombination as by mutation. The long-term mutation rate over millennia is 5–17-fold lower, partly due to the removal of non-synonymous mutations due to purifying selection. Comparisons with the recent literature show that short-term mutation rates vary dramatically in different bacterial species and can span a range of several orders of magnitude

Continuous formal verification of amazon s2n

We describe formal verification of s2n, the open source TLS implementation used in numerous Amazon services. A key aspect of this proof infrastructure is continuous checking, to ensure that properties remain proven during the lifetime of the software. At each change to the code, proofs are automatically re-established with little to no interaction from the developers. We describe the proof itself and the technical decisions that enabled integration into development

Differential regulation of amidase- and formamidase-mediated ammonia production by the Helicobacter pylori fur repressor.

The production of high levels of ammonia allows the human gastric pathogen Helicobacter pylori to survive the acidic conditions in the human stomach. H. pylori produces ammonia through urease-mediated degradation of urea, but it is also able to convert a range of amide substrates into ammonia via its AmiE amidase and AmiF formamidase enzymes. Here data are provided that demonstrate that the iron-responsive regulatory protein Fur directly and indirectly regulates the activity of the two H. pylori amidases. In contrast to other amidase-positive bacteria, amidase and formamidase enzyme activities were not induced by medium supplementation with their respective substrates, acrylamide and formamide. AmiE protein expression and amidase enzyme activity were iron-repressed in H. pylori 26695 but constitutive in the isogenic fur mutant. This regulation was mediated at the transcriptional level via the binding of Fur to the amiE promoter region. In contrast, formamidase enzyme activity was not iron-repressed but was significantly higher in the fur mutant. This effect was not mediated at the transcriptional level, and Fur did not bind to the amiF promoter region. These roles of Fur in regulation of the H. pylori amidases suggest that the H. pylori Fur regulator may have acquired extra functions to compensate for the absence of other regulatory systems

Efficiency of Purine Utilization by Helicobacter pylori: Roles for Adenosine Deaminase and a NupC Homolog

The ability to synthesize and salvage purines is crucial for colonization by a variety of human bacterial pathogens. Helicobacter pylori colonizes the gastric epithelium of humans, yet its specific purine requirements are poorly understood, and the transport mechanisms underlying purine uptake remain unknown. Using a fully defined synthetic growth medium, we determined that H. pylori 26695 possesses a complete salvage pathway that allows for growth on any biological purine nucleobase or nucleoside with the exception of xanthosine. Doubling times in this medium varied between 7 and 14 hours depending on the purine source, with hypoxanthine, inosine and adenosine representing the purines utilized most efficiently for growth. The ability to grow on adenine or adenosine was studied using enzyme assays, revealing deamination of adenosine but not adenine by H. pylori 26695 cell lysates. Using mutant analysis we show that a strain lacking the gene encoding a NupC homolog (HP1180) was growth-retarded in a defined medium supplemented with certain purines. This strain was attenuated for uptake of radiolabeled adenosine, guanosine, and inosine, showing a role for this transporter in uptake of purine nucleosides. Deletion of the GMP biosynthesis gene guaA had no discernible effect on mouse stomach colonization, in contrast to findings in numerous bacterial pathogens. In this study we define a more comprehensive model for purine acquisition and salvage in H. pylori that includes purine uptake by a NupC homolog and catabolism of adenosine via adenosine deaminase