A Longitudinal Study of Bacteriophages as Indicators of Norovirus Contamination of Mussels (Mytilus edulis) and Their Overlying Waters

Sewage pollution leads to the contamination of bivalve shellfish by pathogenic microorganisms. Bacterial indicators support the management of risks associated with the consumption of shellfish; however, they often fail to indicate adequately the potential hazard to human health posed by certain human enteric viruses. Bacteriophages have been proposed as alternative indicators that may more effectively predict the presence of enteric viral pathogens. This study explored the relationships between bacterial indicators (Escherichia coli (E. coli), faecal coliforms (FC) and intestinal enterococci (IE)), phages (somatic (SOMPH), F-specific RNA (F + PH) and human-specific Bacteroides GB-124 phages (GB124PH)) and Norovirus (NoV) (GI/GII) in mussels (Mytilus edulis) and their overlying waters. The bioaccumulation of these indicators and Norovirus in shellfish matrices (e.g., flesh, digestive gland) was investigated bimonthly over a 12-month period in an English estuary. The findings revealed a marked seasonality in the distribution of all organisms, with the highest levels occurring during the autumn/winter months. The levels of all phages in shellfish and their overlying waters correlated better with the levels of Norovirus than with those of bacterial indicators. Somatic coliphages were the indicator that exhibited the strongest correlations with NoV (rho = 0.929). This study suggests that relatively low-cost culture-based phage enumeration appears to offer a more accurate indication of the likely presence of Norovirus in mussels than traditional bacterial indicators

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Insights into the Spectrum of Activity and Mechanism of Action of MGB-BP-3

MGB-BP-3 is a potential first-in-class antibiotic, a Strathclyde Minor Groove Binder (S-MGB), that has successfully completed Phase IIa clinical trials for the treatment of Clostridioides difficile associated disease. Its precise mechanism of action and the origin of limited activity against Gram-negative pathogens are relatively unknown. Herein, treatment with MGB-BP-3 alone significantly inhibited the bacterial growth of the Gram-positive, but not Gram-negative, bacteria as expected. Synergy assays revealed that inefficient intracellular accumulation, through both permeation and efflux, is the likely reason for lack of Gram-negative activity. MGB-BP-3 has strong interactions with its intracellular target, DNA, in both Gram-negative and Gram-positive bacteria, revealed through ultraviolet–visible (UV–vis) thermal melting and fluorescence intercalator displacement assays. MGB-BP-3 was confirmed to bind to dsDNA as a dimer using nano-electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Type II bacterial topoisomerase inhibition assays revealed that MGB-BP-3 was able to interfere with the supercoiling action of gyrase and the relaxation and decatenation actions of topoisomerase IV of both Staphylococcus aureus and Escherichia coli. However, no evidence of stabilization of the cleavage complexes was observed, such as for fluoroquinolones, confirmed by a lack of induction of DSBs and the SOS response in E. coli reporter strains. These results highlight additional mechanisms of action of MGB-BP-3, including interference of the action of type II bacterial topoisomerases. While MGB-BP-3′s lack of Gram-negative activity was confirmed, and an understanding of this presented, the recognition that MGB-BP-3 can target DNA of Gram-negative organisms will enable further iterations of design to achieve a Gram-negative active S-MGB

The Public Health Implications of Real Time PCR Detection of Norovirus in Bivalve Molluscan Shellfish.

Norovirus (NoV) cannot be routinely cultured in the laboratory and therefore existing methods for detection are based on molecular assays. Application of these assays has shown that a high percentage of bivalve molluscan shellfish (BMS) production areas in the UK are contaminated with NoV. It is unclear whether detection of viral genome always correlates with presence of whole infectious virus and thus public health risk. This project aimed to investigate the significance of detecting norovirus in the environment and in BMS by real-time PCR. Murine norovirus (MNV), a closely related NoV, was used as a surrogate in experimental work to determine the behaviour of NoV in the environment and in the BMS matrix in terms of viability and infectivity. A real-time PCR assay for MNV was developed and characterised, and was determined to be equivalent to the in-house real-time PCR assay for human NoV at this laboratory. This assay was then used alongside an existing cell culture method for MNV that was adopted and optimised in this project. The development of a plaque assay with both sufficient robustness and sensitivity for application to bioaccumulated oysters was unsuccessful. These methods, and existing methods for human NoV were used to evaluate environmental persistence of the human NoV genome, human NoV and its surrogate in seawater under artificial environmental conditions simulating winter in the United Kingdom (U. K). The ability of Pacific oysters (Crassostrea gigas) to bioaccumulate both untreated and heat treated NoV particles (from human faeces) and RNA fragments was compared using real-time PCR. Finally, the potential application and incorporation of a pre-extraction treatment into the BMS testing method was assessed. The results presented in this project indicated that NoV RNA may reside in seawater for up to 2 weeks, and that there was no significant difference between the rates of degradation in seawater of naked RNA transcripts and NoV particles from faecal material. However MNV-1 persisted longer in seawater than human NoV under the same conditions. The results presented also indicated that BMS bioaccumulate NoV RNA fragments signifcantly less efficiently than NoV particles from faecal material. Furthermore, the uptake of untreated human NoV and MNV occurred at similar rates to the uptake of heat-treated viruses. It was also determined that using RNaseONE as a pre-extraction treatment to remove PCR signal from ‘free’ RNA and damaged viruses provided scope for the inclusion into the BMS testing method to provide a better prediction of human health risk, however further work would be required to demonstrate its efficacy in a standardised method

The Public Health Implications of Real Time PCR Detection of Norovirus in Bivalve Molluscan Shellfish.

Norovirus (NoV) cannot be routinely cultured in the laboratory and therefore existing methods for detection are based on molecular assays. Application of these assays has shown that a high percentage of bivalve molluscan shellfish (BMS) production areas in the UK are contaminated with NoV. It is unclear whether detection of viral genome always correlates with presence of whole infectious virus and thus public health risk. This project aimed to investigate the significance of detecting norovirus in the environment and in BMS by real-time PCR. Murine norovirus (MNV), a closely related NoV, was used as a surrogate in experimental work to determine the behaviour of NoV in the environment and in the BMS matrix in terms of viability and infectivity. A real-time PCR assay for MNV was developed and characterised, and was determined to be equivalent to the in-house real-time PCR assay for human NoV at this laboratory. This assay was then used alongside an existing cell culture method for MNV that was adopted and optimised in this project. The development of a plaque assay with both sufficient robustness and sensitivity for application to bioaccumulated oysters was unsuccessful. These methods, and existing methods for human NoV were used to evaluate environmental persistence of the human NoV genome, human NoV and its surrogate in seawater under artificial environmental conditions simulating winter in the United Kingdom (U. K). The ability of Pacific oysters (Crassostrea gigas) to bioaccumulate both untreated and heat treated NoV particles (from human faeces) and RNA fragments was compared using real-time PCR. Finally, the potential application and incorporation of a pre-extraction treatment into the BMS testing method was assessed. The results presented in this project indicated that NoV RNA may reside in seawater for up to 2 weeks, and that there was no significant difference between the rates of degradation in seawater of naked RNA transcripts and NoV particles from faecal material. However MNV-1 persisted longer in seawater than human NoV under the same conditions. The results presented also indicated that BMS bioaccumulate NoV RNA fragments signifcantly less efficiently than NoV particles from faecal material. Furthermore, the uptake of untreated human NoV and MNV occurred at similar rates to the uptake of heat-treated viruses. It was also determined that using RNaseONE as a pre-extraction treatment to remove PCR signal from ‘free’ RNA and damaged viruses provided scope for the inclusion into the BMS testing method to provide a better prediction of human health risk, however further work would be required to demonstrate its efficacy in a standardised method

Measuring experimental cyclohexane-water distribution coefficients for the SAMPL5 challenge.

Small molecule distribution coefficients between immiscible nonaqueuous and aqueous phases-such as cyclohexane and water-measure the degree to which small molecules prefer one phase over another at a given pH. As distribution coefficients capture both thermodynamic effects (the free energy of transfer between phases) and chemical effects (protonation state and tautomer effects in aqueous solution), they provide an exacting test of the thermodynamic and chemical accuracy of physical models without the long correlation times inherent to the prediction of more complex properties of relevance to drug discovery, such as protein-ligand binding affinities. For the SAMPL5 challenge, we carried out a blind prediction exercise in which participants were tasked with the prediction of distribution coefficients to assess its potential as a new route for the evaluation and systematic improvement of predictive physical models. These measurements are typically performed for octanol-water, but we opted to utilize cyclohexane for the nonpolar phase. Cyclohexane was suggested to avoid issues with the high water content and persistent heterogeneous structure of water-saturated octanol phases, since it has greatly reduced water content and a homogeneous liquid structure. Using a modified shake-flask LC-MS/MS protocol, we collected cyclohexane/water distribution coefficients for a set of 53 druglike compounds at pH 7.4. These measurements were used as the basis for the SAMPL5 Distribution Coefficient Challenge, where 18 research groups predicted these measurements before the experimental values reported here were released. In this work, we describe the experimental protocol we utilized for measurement of cyclohexane-water distribution coefficients, report the measured data, propose a new bootstrap-based data analysis procedure to incorporate multiple sources of experimental error, and provide insights to help guide future iterations of this valuable exercise in predictive modeling