Abstract

Aims

Cheap, rapid tools for measuring emissions of Plasmopara viticola sporangia directly in the field are required to protect grapevines efficiently and sustainably against downy mildew. To this end, we adapted an existing loop-mediated isothermal amplification (LAMP) protocol based on ITS2 sequences, coupled with a rotating-arm sampler and simple cell lysis, for the in-field measurement of airborne sporangia of P. viticola.

Methods and Results

We estimated the sensitivity and specificity of the molecular reaction with an unpurified DNA template in controlled conditions, using the droplet digital PCR (ddPCR) as a reference. We show that the LAMP lower limit of quantification is 3.3 sporangia.m⁻³ air sampled. Cell lysis in KOH solution was less efficient than CTAB for DNA extraction, but the repeatability of the method was good. We tested this protocol directly in a plot at Chateau Dillon (Blanquefort, France) in which we monitored P. viticola sporangia concentrations from March to October 2020 (88 samples which revealed concentrations ranging from 0 to 243 sporangia.m⁻³). There was a significant quantitative correlation (R ² = 0.52) between ddPCR and LAMP results.

Conclusion

LAMP analysis of an unpurified DNA matrix is a simple and reliable method for in-field estimations of the concentration of airborne P. viticola sporangia.

Significance and Impact of the Study

This study constitutes a first step towards the development of a regional grapevine downy mildew monitoring network in the vineyards of Bordeaux.

Abstract

Aims

Norovirus remains the most significant virological risk that is transmitted via food and the environment to cause acute gastroenteritis. This study aimed to investigate the hypothesis that the contamination of the commercial food production environment with norovirus will be higher in premises that have recently reported a foodborne norovirus outbreak than those that have not.

Methods

Sampling of commercial food production environments was carried out across a 16-month period between January 2015 and April 2016 in the South East and the North West of England by local authority environmental health departments as part of routine surveillance visits to premises. A total of 2982 samples, 2038 virological and 944 bacteriological, were collected from 256 premises. Sixteen of these premises, six from South East and ten from North West England, were sampled as part of a public health outbreak investigation.

Results & Conclusions

Overall, 2038 swabs were submitted for norovirus testing, with an average of eight swabs per premises (range 4 to 23) and a median of seven. Of the premises sampled, 11.7% (30/256) yielded at least one norovirus-positive sample (environmental, and/or food handler hand swab), and 2.5% of the swabs were positive for norovirus. A peak in the positivity rate was seen in the South East in April 2016. No associations were found between norovirus positivity and bacteriology indicators, or between bacteriology indicators and hygiene ratings.

Significance and impact of study

This study demonstrates that food premises and food handlers remain a potential source of norovirus transmission and outbreaks.

Abstract

Aims

To estimate the risk of human rotavirus (RV) and astrovirus (HAstV) infections for swimmers and fishers at Las Cañas beach, Uruguay.

Methods and Results

Surface water samples were collected monthly for 1 year. The dose–response models used were β-Poisson and ₁F₁ hypergeometric for RV and HAstV, respectively. The probabilities of infection were calculated using a kernel density estimate to fitting the data and then sampling from this distribution (Monte Carlo simulation). The probability of RV infection for fishers was between 0 and 65% and for swimmers was between 0 and 50% (<18 years old) and between 0 and 38% (>18 years old). For HAstV, the probability of infection for fishers was between 0% and 45% and for swimmers was between 0 and 38% (<18 years old) and between 0 and 18% (>18 years old).

Conclusions

This study suggests that fishers are at higher risk of infection for both viruses compared with swimmers mainly due to higher viral frequency and concentration at the site for fishing activities.

Abstract

Aims

This study aimed to isolate active substances from metabolites of Bacillus amyloliquefaciens SJ100001 and examine their antifungal activity against Fusarium oxysporum (F. oxysporum) SJ300024 screened from the root–soil of cucumber wilt.

Methods and Results

An active substance, anti-SJ300024, was obtained from the fermentation broth of strain SJ100001 by reversed-phase silica gel and gel chromatography, and further got its chemical structure as cyclic lipopeptide Epichlicin through nuclear magnetic resonance (NMR) and mass spectrometry (MS). In vitro experiments showed that Epichlicin had a better inhibitory rate (67.46%) against the strain SJ300024 than the commercially available fungicide hymexazol (45.10%) at the same concentration. The MTT assays proved that Epichlicin was non-cytotoxic, besides it also had good free radical scavenging ability and total reducing ability.

Conclusions

Epichlicin isolated from strain SJ100001 can effectively control F. oxysporum SJ300024 screened from the root–soil of cucumber wilt.

Significance and Impact of the Study

Epichlicin may be used as an environmentally friendly and efficient biocontrol agent for controlling Fusarium wilt of cucumber and reducing crop losses. More importantly, the non-cytotoxicity of Epichlicin can avoid harm to consumers. Additionally, Epichlicin has broad application prospects in medicine due to its antioxidant properties.

Abstract

Aim

To assess removal versus kill efficacies of antimicrobial treatments against thick biofilms with statistical confidence.

Methods and results

A photo-activated chlorine dioxide treatment (Photo ClO₂) was tested in two independent experiments against thick (>100 μm) Pseudomonas aeruginosa biofilms. Kill efficacy was assessed by viable plate counts. Removal efficacy was assessed by 3D confocal scanning laser microscope imaging (CSLM). Biovolumes were calculated using an image analysis approach that models the penetration limitation of the laser into thick biofilms using Beer's Law. Error bars are provided that account for the spatial correlation of the biofilm's surface. The responsiveness of the biovolumes and plate counts to the increasing contact time of Photo ClO₂ were quite different, with a massive 7 log reduction in viable cells (95% confidence interval [CI]: 6.2, 7.9) but a more moderate 73% reduction in biovolume (95% CI: [60%, 100%]). Results are leveraged to quantitatively assess candidate CSLM experimental designs of thick biofilms.

Conclusions

Photo ClO₂ kills biofilm bacteria but only partially removes the biofilm from the surface. To maximize statistical confidence in assessing removal, imaging experiments should use fewer pixels in each z-slice, and more importantly, at least two independent experiments even if there is only a single field of view in each experiment.

Significance and impact of study

There is limited penetration depth when collecting 3D confocal images of thick biofilms. Removal can be assessed by optimally fitting Beer's Law to all of the intensities in a 3D image and by accounting for the spatial correlation of the biofilm's surface. For thick biofilms, other image analysis approaches are biased or do not provide error bars. We generate unbiased estimates of removal and assess candidate CSLM experimental designs of thick biofilms with different pixilations, numbers of fields of view and number of experiments using the included design tool.

Abstract

Aims

Decomposition, a complicated process, depends on several factors, including carrion insects, bacteria and the environment. However, the composition of and variation in oral bacteria over long periods of decomposition remain unclear. The current study aims to illustrate the composition of oral bacteria and construct an informative model for estimating the post-mortem interval (PMI) during decomposition.

Methods and Results

Samples were collected from rats' oral cavities for 59 days, and 12 time points in the PMI were selected to detect bacterial community structure by sequencing the V3–V4 region of the bacterial 16S ribosomal RNA (16S rRNA) gene on the Ion S5 XL platform. The results indicated that microorganisms in the oral cavity underwent great changes during decomposition, with a tendency for variation to first decrease and then increase at day 24. Additionally, to predict the PMI, an informative model was established using the random forest algorithm. Three genera of bacteria (Atopostipes, Facklamia and Cerasibacillus) were linearly correlated at all 12 time points in the 59-day period. Planococcaceae was selected as the best feature for the last 6 time points. The R ² of the model reached 93.94%, which suggested high predictive accuracy. Furthermore, to predict the functions of the oral microbiota, PICRUSt results showed that energy metabolism was increased on day 3 post-mortem and carbohydrate metabolism surged significantly on days 3 and 24 post-mortem.

Conclusions

Overall, our results suggested that post-mortem oral microbial community data can serve as a forensic resource to estimate the PMI over long time periods.

Significance and Impact of the Study

The results of the present study are beneficial for estimating the PMI. Identifying changes in the bacterial community is of great significance for further understanding the applicability of oral flora in forensic medicine.

Abstract

Aims

We examined the effects of a mixture of contaminants found in agricultural watersheds on the gut microbiota and physiology of both the freshwater mussel Lampsilis cardium, and L. cardium host fish Micropterus salmoides.

Methods and results

Lampsilis cardium and M. salmoides were exposed to three concentrations of agricultural contaminants for 60 days (observing behaviour daily) before being sampled for gut microbiota analyses. DNA was extracted from the gut samples, amplified via PCR, and sequenced using the Illumina Mi-Seq platform. Only L. cardium guts had differing microbiota across treatments, with an increase in potentially pathogenic Aeromonas. We also provide novel evidence of a core microbiota within L. cardium and M. salmoides. In terms of physiology, female L. cardium exhibited a decrease in movement and marsupial gill display in contaminant exposures.

Conclusions

Exposure to contaminants from agricultural watersheds may affect population recruitment within freshwater mussel communities over time. Specifically, increased pathogenic micro-organisms and altered behaviour can reduce the likelihood of glochidia dispersal.

Significance and impact of the study

This study supports emerging research that contaminants found in agricultural watersheds may be a factor in freshwater mussel population declines. It also provides novel evidence that unionids have a core gut microbiota.

Abstract

Aim

Polymicrobial biofilm encasing cross-kingdom micro-organisms are apparent in medicine, which imposes serious resistance to conventional antimicrobial treatment. The objective of the study was to explore Butea monosperma seed lectin (BMSL) conjugated antimicrobial lipid, 2-((N-[2-hydroxyethyl]palmitamido)methyl)-1-methylpyridin-1-ium iodide (cN16E) to inhibit mixed-species biofilm of uropathogenic Escherichia coli–Candida albicans.

Methods and Results

Antimicrobial activity and antibiofilm of cN16E and cN16E-BMSL conjugate (BcN16E) were analysed against single- and mixed microbial cultures. The minimum inhibitory concentration (MIC) indicates that the MIC of cN16E-BMSL conjugate (BcN16E) against cohabiting UPEC-C. albicans was eightfold lower than the cN16E. BcN16E affects membrane integrity to elicit antimicrobial activity. BcN16E inhibits the dual-species biofilm even with 16 times lower MIC of cN16E. BcN16E impairs the biofilm-associated virulence factors which include extracellular polysaccharides, cell surface hydrophobicity, swimming, swarming motilities, hyphal filamentous morphology, curli formation and haemolysin activity. As a proof of concept, we demonstrated BcN16E ability to inhibit dual-species biofilm formation on a urinary catheter.

Conclusion

The study revealed that the BcN16E is better than cN16E in impairing biofilm-associated virulence factors and exerting antimicrobial activity.

Significance and Impact of the Study

The findings emphasize that phytolectin has the potential to enhance the anti-virulence strategies of antimicrobials against cross-kingdom biofilm-related infections.