Category Archives: ORIGINAL ARTICLE

Imaging and plate counting to quantify the effect of an antimicrobial: A case study of a photo‐activated chlorine dioxide treatment

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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.

Extraction and characterization of cyclic lipopeptides with antifungal and antioxidant activities from Bacillus amyloliquefaciens

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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.

Comparison of the risk of infection of human rotavirus and astrovirus according to fishing and swimming activities at Las Cañas beach, Uruguay

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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.

Norovirus attribution study: Detection of norovirus from the commercial food preparation environment in outbreak and non‐outbreak premises

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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.

LAMP for in‐field quantitative assessments of airborne grapevine downy mildew inoculum

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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.

Natamycin as a safe food additive to control postharvest green mould and sour rot in citrus

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Abstract

Aims

The purpose of this study was to explore the potential inhibitory mechanism and assess the feasibility of natamycin as an antifungal agent in the utilization of citrus storage.

Methods and Results

In this study, the mycelial growth, spore germination as well as germ tube elongations of Geotrichum citri-aurantii and Penicillium digitatum were significantly inhibited by natamycin treatment. The relative conductivities of G. citri-aurantii and P. digitatum mycelia were increased as time went by and the damages of plasma membranes were up to 17.43% and 28.61%. The mitochondria abnormalities and vacuolation were also observed in the TEM. Moreover, the sour rot and green mould decay incidences were reduced to 18.33% and 10% post incubation with G. citri-aurantii and P. digitatum under 300 mg L−1 natamycin application, respectively. For the citrus storage experiment, there was no significant difference in edible rate, juice yield, total soluble solid (TSS) content, titratable acid (TA) and decay incidences of the ‘Newhall’ navel orange fruit treated with 300 mg L−1 natamycin stored for 90 d.

Conclusions

Natamycin could decrease the expansions of green mould and sour rot and maintain quality and improve storability on citrus fruit.

Significance and Impact of the study

This work explores the potential inhibition mechanism of natamycin G. citri-aurantii and P. digitatum and assesses the feasibility of natamycin as an antifungal agent in the utilization of citrus storage.

Isolation, purification and structural elucidation of Mellein from endophytic fungus Lasiodiplodia theobromae strain (SJF‐1) and its broad‐spectrum antimicrobial and pharmacological properties

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Isolation, purification and structural elucidation of Mellein from endophytic fungus Lasiodiplodia theobromae strain (SJF-1) and its broad-spectrum antimicrobial and pharmacological properties

Significance and Impact of the Study: In the present study, the bioactive metabolite Mellein was represented as a broad-spectrum antimicrobial agent produced by Lasiodiplodia theobromae strain (SJF-1) isolated from the medicinal plant Syzygium cumini. In addition, Mellein exhibited drug-like characteristics through in silico absorption, distribution, metabolism, and excretion pharmacological studies. Hence, from the above studies, Mellein could be considered as a potential drug candidate and also a biocontrol agent in the medical, agricultural and pharmaceutical sectors.


Abstract

In an on-going investigation of bioactive metabolites producing potential endophytic fungi, the strain Lasiodiplodia theobromae (SJF-1) was isolated from a medicinal plant Syzygium cumini. The cultural, morphological and molecular identification was done with the SJF-1 strain. The obtained gene sequence was deposited in NCBI with accession number MG 938644. The methanolic extract of SJF-1 strain possessed one major bioactive fraction, and it was purified by column chromatography. Further, it was identified as Mellein by various spectroscopic studies (1H, 13C, DEPT-135°, FT-IR, ESI-HR-MS and 2D NMR). Biologically, Mellein showed potent anti-Xanthomonas activity with minimum inhibitory concentration (MIC) values ranging from 1·9 to 62·5 μg ml−1 against 11 Xanthomonas strains, a broad-spectrum antimicrobial activity with MIC 7·8–31·25 μg ml−1 and 1·9–31·25 μg ml−1 towards both bacterial and fungal strains, respectively. The scanning electron microscope analysis proved the antimicrobial efficacy of a Mellein by rupturing the cell walls of Xanthomonas sp. Molecular docking studies further supported that the Mellein showed good binding interactions with the proteins of Xanthomonas sp. to reduce pathogenicity. Further, in silico pharmacological studies showed that this metabolite exhibited high gastrointestinal absorption properties and promising oral drug bioavailability. We report, anti-Xanthomonas, in silico docking and pharmacological studies of Mellein from (SJF-1) strain for the first time.

Biodeterioration of low‐density polyethylene by mangrove‐associated endolichenic fungi and their enzymatic regimes

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Biodeterioration of low-density polyethylene by mangrove-associated endolichenic fungi and their enzymatic regimes

Significance and Impact of the Study: Endolichenic fungi (ELF) are a unique group of organisms with the ability to sustain under challenging conditions and have rich and versatile metabolite profiles. The present study showed that several ELF, isolated from mangrove ecosystems could grow in low-density polyethylene (LDPE) amended media and showed signatures of LDPE biodeterioration. Qualitative and quantitative enzymatic assays of six extracellular fungal enzymes, having roles in polymer depolymerization were also performed. Results highlighted that six ELF species rich in enzymatic profiles were capable of LDPE biodeterioration.


Abstract

Fungal involvement in the biodeterioration of low-density polyethylene (LDPE) has received great attention in recent years. Among diverse groups of fungi, endolichenic fungi (ELF) are adapted to thrive in resource-limited conditions. The present study was designed to investigate the potential of mangrove-associated ELF, in the biodeterioration of LDPE and to quantify key-depolymerizing enzymes. A total of 31 ELF species, isolated from 22 lichens of mangrove ecosystems in Negombo lagoon, Sri Lanka were identified using DNA barcoding techniques. ELF were inoculated into a mineral salt medium, containing LDPE strips and incubated at 28 ± 2°C, for 21 days, under laboratory conditions. After incubation, biodeterioration was monitored based on percent reductions in weights and tensile properties, increments in the degree of water absorption, changes in peaks of infrared spectra and surface erosions using scanning electron microscopy. Out of 31 species, Chaetomium globosum, Daldinia eschscholtzii, Neofusicoccum occulatum, Phanerochaete chrysosporium, Schizophyllum commune and Xylaria feejeensis showed significant changes. Production of depolymerizing enzymes by these species was assayed qualitatively using plate-based methods and quantitatively by mass-level enzyme production. Among them, Phanerochaete chrysosporium showed the highest enzyme activities as (9·69 ± 0·04) × 10−3, (1·96 ± 0·01) × 10−3, (5·73 ± 0·03) × 10−3, (0·88 ± 0·01), (0·64 ± 0·06), (1·43 ± 0·01) U ml−1 for laccase, lignin peroxidase, manganese peroxidase, amylase, lipase and esterase, respectively.

ERIC‐PCR‐based molecular typing of multidrug‐resistant Escherichia coli isolated from houseflies (Musca domestica) in the environment of milk and meat shops

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ERIC-PCR-based molecular typing of multidrug-resistant Escherichia coli isolated from houseflies (Musca domestica) in the environment of milk and meat shops

Significance and impact of the study: Houseflies (Musca domestica) are endophilic cosmopolitan pests of medical and veterinary importance. These are associated with regions of human activities such as food centres, butcher houses, animal farms and surrounding areas and create an annoyance to people, poultry and domestic farm animals. The present study reports that housefly samples collected from milk and meat outlets were positive for multidrug-resistant (MDR) E. coli and harbour bla CTX-M and bla TEM genes in plasmid along with other resistant genes for tetracyclines and quinolones. The presence of MDR genes in bacteria isolated from housefly samples is of serious concern from public health and food safety points of view. Investigation of genetic determinants of MDR in bacteria would be of great value for formulating and validating suitable control strategies to combat the spread of antimicrobial resistance.


Abstract

The emergence and spread of antimicrobial resistance have become a major global public health concern. A component of this problem is the spread of antibiotic-resistant bacteria. Flies move freely between habitats of food-producing animals and human beings and thus have great potential for dissemination of antimicrobial-resistant bacteria from a contaminated environment to milk and meat markets, posing potential hazards for consumers. During the present study, a total of 150 houseflies were captured from milk and meat shops located in Durg and Raipur city of Chhattisgarh, India. The Escherichia coli were isolated from houseflies and characterized on the basis of cultural and molecular tests. Further, the isolates were subjected to antimicrobial susceptibility testing against frequently used antibiotics using the disk diffusion method. The antibiotic resistance genes and int1 gene were detected using polymerase chain reaction (PCR). A total of 45 E. coli isolates were obtained from the fly samples with an overall prevalence rate of 30·0%. Antibiogram results confirmed that E. coli isolates were resistant to multiple antibiotics. Out of the (45) isolates of E. coli, 17 (37·8%) isolates were extended-spectrum beta-lactamase (ESBL) producer and multi-drug-resistant (MDR). Out of the ESBL and MDR E. coli isolates, bla CTX-M (24·4%), bla TEM (11·1%), tetA (28·8%), tetB (26·7%), gyrA (26·7%), parC (31. 1%) and int1 genes (15·5%) were detected but none of the isolates were found positive for bla SHV gene. Findings of the present study confirm that MDR E. coli are widely distributed in houseflies and play an important role in the transmission of antibiotic-resistant bacteria from contaminated environments to milk and meat shop environment.

Solid‐state fermentation produces greater stearidonic acid levels in genetically engineered Mucor circinelloides

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Solid-state fermentation produces greater stearidonic acid levels in genetically engineered Mucor circinelloides

Significance and Impact of the Study: Solid-state fermentation is an efficient and economical method for lipid accumulation in the oleaginous microorganism. In this study, engineered strains Mucor circinelloides McD15D with overexpressing the delta15-desaturase were able to accumulate more stearidonic acid (SDA) under solid-state fermentation than submerged fermentation, providing a new strategy for fermentation technology to enhance SDA production by oleaginous microorganisms.


Abstract

Omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) are important dietary components due to their health benefits and preventative role in cardiovascular disease. Fish-based and plant seed oils are rich in stearidonic acid (SDA; 18:4, n-3), which are readily metabolized into ω-3 PUFAs such as eicosapentaenoic acid. However, these natural sources of SDA are generally low yielding and are unlikely to meet global demands, so new sustainable microbial fermentative sources of SDA need to be identified. Expression of delta15-desaturase in the oleaginous filamentous fungus Mucor circinelloides (McD15D) has been used to construct a recombinant SDA-producing McD15D strain that produces 5·0% SDA levels using submerged fermentation conditions. Switching to solid-state fermentation conditions in the same medium with submerged fermentation resulted in this engineered strain producing significantly higher amounts of SDA. A Box–Behnken design of response surface methodology approach has been used to identify optimal glucose and ammonium tartrate concentrations and temperature levels to maximize SDA production. The use of these optimal solid-state fermentation conditions resulted in the spores and mycelium of the recombinant McD15D producing 19·5% (0·64 mg g−1) and 12·2% (1·52 mg g−1) SDA content, respectively, which represents an overall increase in SDA yield of 188·0% compared with SDA yields produced using submerged fermentation conditions.