Alejandro Garrido-Maestu

Application, mode of action, and in vivo activity of chitosan and its micro- and nanoparticles as antimicrobial agents: A review

Chitosan is widely used as an antimicrobial agent due to its biodegradability, nontoxicity, and antimicrobial properties. Although in vitro antimicrobial activity of chitosan and its derivatives were reviewed recently, its in vivo activity is not recapitulated sufficiently. This review will focus on recent studies of in vivo antimicrobial activity of chitosan and its micro- and nanoparticles to enhance food safety and animal diseases treatment. Three major factors affecting the antimicrobial activity include microbial, intrinsic, and environmental factors are discussed. The accepted and potential mechanisms regarding the use of chitosan and its micro- and nanoparticles are discussed to further understand their antimicrobial properties.

Detection of foodborne pathogens by qPCR: A practical approach for food industry applications

Abstract Microbiological analysis of food is an integrated part of microbial safety management in the food chain. Monitoring and controlling foodborne pathogens are traditionally carried out by conventional microbiological methods based on culture-dependent approaches in control laboratories and private companies. However, polymerase chain reaction (PCR) has revolutionized microbiological analysis allowing detection of pathogenic microorganisms in food, without the necessity of classical isolation and identification. However, at present, PCR and quantitative polymerase chain reaction (qPCR) are essential analytical tools for researchers working in the field of foodborne pathogens. This manuscript reviews recently described qPCR methods applied for foodborne bacteria detection, serving as economical, safe, and reliable alternatives for application in the food industry and control laboratories. Multiplex qPCR, which allows the simultaneous detection of more than one pathogen in one single reaction, saving considerable effort, time, and money, is emphasized in the article.

lolB gene, a valid alternative for qPCR detection of Vibrio cholerae in food and environmental samples

In recent years a new genetic target for Vibrio cholerae detection has been reported, but its application was limited to clinical samples. This target, lolB, has never been applied to either food or environmental samples. In the present study the development, as well as the evaluation and pre-validation, of a real-time PCR method based on lolB, is described. The method included a newly designed hydrolysis probe to enhance its specificity. After comparison against other molecular and traditional methods, similar results were obtained regarding relative sensitivity, relative specificity, relative accuracy, positive and negative predictive values and index kappa of concordance (all higher than 91%), as well as a very low limit of detection (2 cfu/25 g). Additionally, after the analysis of more than 160 different food and environmental samples, its applicability in the food industry was completely demonstrated.

Re-evaluation of Enhanced qPCR Prevalidated Method for Next-day Detection of Salmonella spp., Shigella spp., Escherichia coli O157 and Listeria monocytogenes

The current study was conducted in order to reduce the overall time needed in a previously validated qPCR method, and extend the procedure for the simultaneous detection of an additional pathogen (Shigella spp.).The original method was modified by extending the primary enrichment and eliminating the secondary enrichment. Additionally, a rapid commercial DNA extraction kit was evaluated against our reference protocol taking into consideration DNA concentration obtained, purity of the DNA extracts evaluating two absorbance ratios (260/280 and 260/230), and Cq and final fluorescence after qPCR analysis. Comparable results were obtained with both DNA extraction methods, but the commercial kit performed worse with low bacterial concentrations. A total of 84 spiked and blind samples were analyzed with the rapid protocol without finding significant differences with respect to the original method regarding qPCR efficiency (90–103%), and all the method’s parameters that were previously analyzed (above 91%). Additionally, a very low limit of detection was still obtained after the method optimization (below 10 cfu/25 g). The modified method represents a significant advance in detection of foodborne pathogens because it can provide accurate and reliable results for producers and health authorities in less than 27 h including the enrichment step. This protocol implementation resulted in an overall 5 h reduction, with less hands-on work, and without any loss in the quality of the method. In addition the inclusion of an additional pathogen extends the method’s applicability to 4 pathogens of interest.

Combination of Microfluidic Loop-Mediated Isothermal Amplification with Gold Nanoparticles for Rapid Detection of Salmonella spp. in Food Samples

Foodborne diseases are an important cause of morbidity and mortality. According to the World Health Organization, there are 31 main global hazards, which caused in 2010 600 million foodborne illnesses and 420000 deaths. Among them, Salmonella spp. is one of the most important human pathogens, accounting for more than 90000 cases in Europe and even more in the United States per year. In the current study we report the development, and thorough evaluation in food samples, of a microfluidic system combining loop-mediated isothermal amplification with gold nanoparticles (AuNPs). This system is intended for low-cost, in situ, detection of different pathogens, as the proposed methodology can be extrapolated to different microorganisms. A very low limit of detection (10 cfu/25 g) was obtained. Furthermore, the evaluation of spiked food samples (chicken, turkey, egg products), completely matched the expected results, as denoted by the index kappa of concordance (value of 1.00). The results obtained for the relative sensitivity, specificity and accuracy were of 100% as well as the positive and negative predictive values.

Comprehensive in vitro and in vivo risk assessments of chitosan microparticles using human epithelial cells and Caenorhabditis elegans

The safety of using nano- and microparticles is a developing concern. In this study, we conducted risk assessments of chitosan microparticles (CMs) using in vitro human epithelial cell lines and in vivo animal model, Caenorhabditis elegans. After engineering of various CMs, we screened four CMs based on antimicrobial activity, which is a potential usage for disease treatment caused by multidrug resistant bacteria, and evaluated for risk assessments. CMs, with strong antimicrobial activity, and inorganic nanoparticles (SiO2, TiO2, and ZnO) did not cause toxicity in human cells measured by cell membrane integrity, mitochondria activity, and reactive oxygen species concentration. However, when applied to C. elegans, only CMs generated with low molecular weight chitosan and tripolyphosphate at 0.1% did not affect the lifespan, while the other CMs and inorganic nanoparticles shortened the lifespan, suggesting that they may cause subtle toxicity. These results suggest that C. elegans could be a sensitive animal model to measure low level of toxicity of nano- and microparticles. Taken together, although CMs do not cause toxicity at working concentrations of antimicrobial activity in human epithelial cells, they may cause toxicity at high concentration, suggesting that nano- and microparicles should be thoroughly investigated before they are applied.

Microbiological Quality of Ready-to-Eat Pickled Fish Products

The microbiological quality of 18 commercially available in Spain ready-to-eat fish products containing Engraulidae was evaluated through application of the corresponding ISO procedures for total mesophilic aerobic microbial counts, detection and enumeration of enterobacteria, and detection of Staphylococcus spp. All isolates were identified to the species level using two different biochemical methods: the API® test and the Biolog® identification system. The most commonly occurring contaminants found were Enterobacteriaceae—such as Citrobacter freundii and other Citrobacter species, Enterobacter cloacae, Cronobacter sakazakii, Hafnia alvei, Pantoea, Proteus ssp., and Escherichia coli. The presence of such opportunistic pathogens and contaminant microflora was confirmed in 61% of the foods sampled.

Application of real-time PCR for early diagnosis of diseases caused by Aeromonas salmonicida, Vibrio anguillarum, and Tenacibaculum maritimum in turbot: A field study

ABSTRACT In the present study a multiplex real-time PCR method was developed for early detection of diseased fish infected by Aeromonas salmonicida, Vibrio anguillarum, and/or Tenacibaculum maritimum. The method consisted of the detection of three species-specific genes after DNA extraction with a commercial kit. Three types of samples were tested, and the results were compared with those of traditional diagnosis. The method obtained a limit of detection of 104 cfu/mL (2 x 102 cfu/tube). Additionally, 27 samples from fish showing signs of disease were correctly diagnosed by the developed methodology, demonstrating its suitability for implementation in aquaculture.

Highly sensitive detection of gluten-containing cereals in food samples by real-time Loop-mediated isothermal AMPlification (qLAMP) and real-time polymerase chain reaction (qPCR)

The treatment of gluten-related disorders is based on a lifelong, and strict, gluten-free diet. Thus, reliable and sensitive methods are required to detect the presence of gluten contamination. Traditional techniques rely on the detection of these proteins based on specific antibodies, but recent approaches go for an indirect route detecting the DNA that indicates the presence of cereals with gluten content. In the current study two different DNA amplification techniques, real-time PCR (qPCR) and real-time Loop-mediated isothermal AMPlification (qLAMP), were evaluated for their capability to detect and quantify gluten. Different detection strategies, based on these DNA amplification techniques, were tested. Even though good specificity results were obtained with the different approaches, overall qPCR proved more sensitive than qLAMP. This is the first study reporting a qLAMP based-method for the detection of gluten-containing cereals, along with its evaluation in comparison with qPCR.

Data on minute DNA quantification on microvolumetric solutions: comparison of mathematical models and effect of some compounds on the DNA quantification accuracy

This article contains data related to the research article entitled “Novel approach for accurate minute DNA quantification on microvolumetric solutions” (Carvalho et al., 2018). The combination of PicoGreen® with a microvolume fluorospectrometer is a popular DNA quantification method due to its high sensitivity and minimal consumption of sample, being commonly used to evaluate the performance of microfluidic devices designed for DNA purification. In this study, the authors present data related with the effect of DNA fragmentation level. The present data article includes the data used on the precision evaluation, in terms of repeatability, of the mathematical models developed to obtain the standards curve for salmon sperm DNA (low molecular weight). In addition, results related with the effect of some compounds on the DNA quantification accuracy using λDNA are presented.