Daniel Berdejo
@university of zaragoza
Department of Animal Production and Food Science, Faculty of Veterinary Medicine
Assistant Professor
RESEARCH, TEACHING, or OTHER INTERESTS
Food Science, Microbiology, Molecular Biology, Health, Toxicology and Mutagenesis
Scopus Publications
Scopus Publications
Elisa Pagan, Natalia Merino, Daniel Berdejo, Raul Campillo, Elisa Gayan, Diego García-Gonzalo, and Rafael Pagan
Springer Science and Business Media LLC
Abstract Emergence of genetic variants with increased resistance/tolerance to natural antimicrobials, such as essential oils, has been previously evidenced; however, it is unknown whether mutagenesis follows a general or a specific pattern. For this purpose, we carried out four adaptive laboratory evolutions (ALE) in parallel of Salmonella enterica Typhimurium with carvacrol. After 10 evolution steps, we selected and characterized one colony from each lineage (SeCarA, SeCarB, SeCarC, and SeCarD). Phenotypic characterization of the four evolved strains revealed enhanced survival to lethal treatments; two of them (SeCarA and SeCarB) showed an increase of minimum inhibitory concentration of carvacrol and a better growth fitness in the presence of carvacrol compared to wild-type strain. Whole genome sequencing revealed 10 mutations, of which four (rrsH, sseG, wbaV, and flhA) were present in more than one strain, whereas six (nirC, fliH, lon, rob, upstream yfhP, and upstream argR) were unique to individual strains. Single-mutation genetic constructs in SeWT confirmed lon and rob as responsible for the increased resistance to carvacrol as well as to antibiotics (ampicillin, ciprofloxacin, chloramphenicol, nalidixic acid, rifampicin, tetracycline, and trimethoprim). wbaV played an important role in increased tolerance against carvacrol and chloramphenicol, and flhA in cross-tolerance to heat treatments. As a conclusion, no common phenotypical or genotypical pattern was observed in the isolated resistant variants of Salmonella Typhimurium emerged under carvacrol stress. Furthermore, the demonstration of cross-resistance against heat and antibiotics exhibited by resistant variants raises concerns regarding food safety. Key points • Stable resistant variants of Salmonella Typhimurium emerged under carvacrol stress • No common pattern of mutagenesis after cyclic exposures to carvacrol was observed • Resistant variants to carvacrol showed cross-resistance to heat and to antibiotics Graphical abstract
Elisa Pagán, Foteini Pavli, Sarah Happiette, Daniel Berdejo, Ruben Gatt, Rafael Pagán, Vasilis Valdramidis, and Diego García-Gonzalo
Elsevier BV
Daniel Berdejo, Julien Mortier, Alexander Cambré, Malgorzata Sobota, Ronald Van Eyken, Tom Dongmin Kim, Kristof Vanoirbeek, Diego García Gonzalo, Rafael Pagán, Médéric Diard,et al.
American Society for Microbiology
ABSTRACT Understanding the evolutionary dynamics of foodborne pathogens throughout our food production chain is of utmost importance. In this study, we reveal that Salmonella Typhimurium can readily and reproducibly acquire vastly increased heat shock resistance upon repeated exposure to heat shock. Counterintuitively, this boost in heat shock resistance was invariantly acquired through loss-of-function mutations in the dnaJ gene, encoding a heat shock protein that acts as a molecular co-chaperone of DnaK and enables its role in protein folding and disaggregation. As a trade-off, however, the acquisition of heat shock resistance inevitably led to attenuated growth at 37°C and higher temperatures. Interestingly, loss of DnaJ also downregulated the activity of the master virulence regulator HilD, thereby lowering the fraction of virulence-expressing cells within the population and attenuating virulence in mice. By connecting heat shock resistance evolution to attenuation of HilD activity, our results confirm the complex interplay between stress resistance and virulence in Salmonella Typhimurium. IMPORTANCE Bacterial pathogens such as Salmonella Typhimurium are equipped with both stress response and virulence features in order to navigate across a variety of complex inhospitable environments that range from food-processing plants up to the gastrointestinal tract of its animal host. In this context, however, it remains obscure whether and how adaptation to one environment would obstruct fitness in another. In this study, we reveal that severe heat stress counterintuitively, but invariantly, led to the selection of S. Typhimurium mutants that are compromised in the activity of the DnaJ heat shock protein. While these mutants obtained massively increased heat resistance, their virulence became greatly attenuated. Our observations, therefore, reveal a delicate balance between optimal tuning of stress response and virulence features in bacterial pathogens.
Natalia Merino, Daniel Berdejo, Elisa Pagán, Claire Girard, Sylvain Kerros, Eleonora Spinozzi, Rafael Pagán, and Diego García-Gonzalo
MDPI AG
On account of the widespread development and propagation of antimicrobial-resistant (AMR) bacteria, essential oils (EOs) have emerged as potential alternatives to antibiotics. However, as already observed for antibiotics, recent studies have raised concerns regarding the potential emergence of resistant variants (RVs) to EOs. In this study, we assessed the emergence of RVs in Escherichia coli and Salmonella enterica Typhimurium after evolution assays under extended exposure to subinhibitory doses of two commercial EOs (AEN and COLIFIT) as well as to two antibiotics (amoxicillin and colistin). Phenotypic characterization of RVs from evolution assays with commercial EOs yielded no relevant increases in the minimum inhibitory concentration (MIC) of E. coli and did not even modify MIC values in S. Typhimurium. Conversely, RVs of E. coli and S. Typhimurium isolated from evolution assays with antibiotics showed increased resistance. Genotypic analysis demonstrated that resistance to commercial EOs was associated with enhanced protection against oxidative stress and redirection of cell energy toward efflux activity, while resistance to antibiotics was primarily linked to modifications in the cell binding sites of antibiotics. These findings suggest that AEN and COLIFIT could serve as safe alternatives to antibiotics in combating the emergence and dissemination of antimicrobial resistance within the agrifood system.
Víctor Calvo, Laura Fuentes, Daniel Berdejo, José M. González-Domínguez, Wolfgang K. Maser, and Ana M. Benito
MDPI AG
Pickering emulsions (PEs) differ from conventional emulsions in the use of solid colloidal particles as stabilizing agents instead of traditional amphiphilic molecules. Nanostructured biopolymers (NBs) emerge as a promising alternative for PE stabilization owing to their remarkable biocompatibility, abundant availability, and low cost. To explore this potential, a study is herein presented, in which cellulose nanocrystals (CNCs), both type I and type II allomorphs, and chitin nanocrystals (ChNCs) were used for stabilizing oil-in-water PEs prepared by the use of ultrasound. Sunflower oil was selected as the oil phase as it offers the advantages of being edible, renewable, and inexpensive. By utilizing ζ-potential, static light diffraction, and visual observations, we determined the optimal oil/water ratio for each type of NB to obtain stable emulsions after 14 days. The optimized PEs were used to form bacterial nanocellulose composites through emulsion templating. To our knowledge, this study represents a pioneering work in exploiting oil-in-water PEs for this approach. Additionally, it entails the first utilization of nonmercerized type II CNCs as stabilizers for PEs, while also establishing a direct comparison among the most relevant NBs. The resulting composites exhibited a unique morphology, composed of larger pores compared to standard bacterial nanocellulose aerogels. These findings highlight the notable potential of NBs as stabilizers for PEs and their ability to generate green nanocomposites with tailored properties.
Daniel Berdejo, Diego García-Gonzalo, Nadia Oulahal, Rositsa Denkova-Kostova, Vesela Shopska, Georgi Kostov, Pascal Degraeve, and Rafael Pagan
Faculty of Food Technology and Biotechnology - University of Zagreb
Tailor-made foods, also known as foods with programmable properties, are specialised systems with unique composition prepared by different methods, using the known mechanisms of action of their bioactive ingredients. The development of tailor-made foods involves the evaluation of individual components, including bioactive substances derived from waste products of other productions, such as essential oils. These components are evaluated both individually and in combination within food compositions to achieve specific functionalities. This review focuses on the application of minimal processing technologies for the production and preservation of tailor-made foods. It examines a range of approaches, including traditional and emerging technologies, as well as novel ingredients such as biomolecules from various sources and microorganisms. These approaches are combined according to the principles of hurdle technology to achieve effective synergistic effects that enhance food safety and extend the shelf life of tailor-made foods, while maintaining their functional properties.
Daniel Berdejo, Elisa Gayán, Elisa Pagán, Natalia Merino, Raúl Campillo, Rafael Pagán, and Diego García-Gonzalo
MDPI AG
Essential oils and their constituents, such as carvacrol, are potential food preservatives because of their great antimicrobial properties. However, the long-term effects of these compounds are unknown and raise the question of whether resistance to these antimicrobials could emerge. This work aims to evaluate the occurrence of genetic resistant variants (RVs) in Listeria monocytogenes EGD-e by exposure to carvacrol. Two protocols were performed for the RVs selection: (a) by continuous exposure to sublethal doses, where LmSCar was isolated, and (b) by reiterative exposure to short lethal treatments of carvacrol, where LmLCar was isolated. Both RVs showed an increase in carvacrol resistance. Moreover, LmLCar revealed an increased cross-resistance to heat treatments at acid conditions and to ampicillin. Whole-genome sequencing identified two single nucleotide variations in LmSCar and three non-silent mutations in LmLCar. Among them, those located in the genes encoding the transcriptional regulators RsbT (in LmSCar) and ManR (in LmLCar) could contribute to their increased carvacrol resistance. These results provide information regarding the mode of action of this antimicrobial and support the importance of knowing how RVs appear. Further studies are required to determine the emergence of RVs in food matrices and their impact on food safety.
Daniel Berdejo, Elisa Pagán, Natalia Merino, Laura Botello-Morte, Rafael Pagán, and Diego García-Gonzalo
Elsevier BV
Daniel Berdejo, Elisa Pagán, Natalia Merino, Diego García-Gonzalo, and Rafael Pagán
Elsevier BV
Santiago López‐Miranda, Daniel Berdejo, Elisa Pagán, Diego García‐Gonzalo, and Rafael Pagán
Wiley
AbstractBACKGROUNDThe antimicrobial activity of essential oils and their constituents has led to increasing interest in using them as natural preservative agents. However, their high sensitivity to light and oxygen, their volatility and their low aqueous solubility are all obstacles to their application in the food, cosmetic or pharmaceutical industries. Encapsulation in cyclodextrins (CDs) is a solution for the application of such essential oils.RESULTSThe complexation of carvacrol and thymol with hydroxypropyl (HP)‐α‐, HP‐β‐ and HP‐γ‐CD, the behavior of the solid complexes prepared by freeze‐drying and spray‐drying methods and the antibacterial activity of solid complexes were studied. Kc values of HP‐α‐ and HP‐γ‐CD complexes with carvacrol (118.4 and 365.7 L mol−1) and thymol (112.5 and 239.7 L mol −1) were far lower than those observed for HP‐β‐CD complexes with carvacrol (2268.2 L mol −1) and thymol (881.6 L mol −1). The lower stability of HP‐α‐ and HP‐γ‐CD complexes increased the release of compounds, thereby affecting the antimicrobial activity of carvacrol and thymol to a lesser extent than complexation with HP‐β‐CD, normally used in the encapsulation of carvacrol and thymol. HP‐β‐CD encapsulation of carvacrol and thymol markedly reduced their antimicrobial activity. The freeze‐drying method barely affected the antimicrobial activity of carvacrol and thymol after encapsulation, while spray drying could be considered for the production of solid complexes in combination with the appropriate CD.CONCLUSIONSIt was thus demonstrated that HP‐α‐ and HP‐γ‐CD are very suitable alternatives for the encapsulation of carvacrol and thymol with the purpose of preserving their bacteriostatic and bactericidal activities. © 2020 Society of Chemical Industry
Roberta Bento, Elisa Pagán, Daniel Berdejo, Rayssa Julliane de Carvalho, Sonia García-Embid, Filippo Maggi, Marciane Magnani, Evandro Leite de Souza, Diego García-Gonzalo, and Rafael Pagán
Elsevier BV
Daniel Berdejo, Elisa Pagán, Natalia Merino, Rafael Pagán, and Diego García-Gonzalo
MDPI AG
Emergence of strains with increased resistance/tolerance to natural antimicrobials was evidenced after cyclic exposure to carvacrol, citral, and (+)-limonene oxide. However, no previous studies have reported the development of resistance and tolerance to complex essential oils (EOs). This study seeks to evaluate the occurrence of Staphylococcus aureus strains resistant and tolerant to a complex orange essential oil (OEO) after prolonged cyclic treatments at low concentrations. Phenotypic characterization of evolved strains revealed an increase of minimum inhibitory and bactericidal concentration for OEO, a better growth fitness in presence of OEO, and an enhanced survival to lethal treatments, compared to wild-type strain. However, no significant differences (p > 0.05) in cross-resistance to antibiotics were observed. Mutations in hepT and accA in evolved strains highlight the important role of oxidative stress in the cell response to OEO, as well as the relevance of the cell membrane in the cell response to these natural antimicrobials. This study demonstrates the emergence of S. aureus strains that are resistant and tolerant to EO (Citrus sinensis). This phenomenon should be taken into account to assure the efficacy of natural antimicrobials in the design of food preservation strategies, in cleaning and disinfection protocols, and in clinical applications against resistant bacteria.
Daniel Berdejo, Natalia Merino, Elisa Pagán, Diego García-Gonzalo, and Rafael Pagán
MDPI AG
The emergence of antimicrobial resistance has raised questions about the safety of essential oils and their individual constituents as food preservatives and as disinfection agents. Further research is required to understand how and under what conditions stable genotypic resistance might occur in food pathogens. Evolution experiments on Salmonella Typhimurium cyclically exposed to sublethal and lethal doses of carvacrol permitted the isolation of SeSCar and SeLCar strains, respectively. Both evolved strains showed a significant increase in carvacrol resistance, assessed by minimum inhibitory and bactericidal concentrations, the study of growth kinetics in the presence of carvacrol, and the evaluation of survival under lethal conditions. Moreover, antibiotic susceptibility tests revealed a development of SeLCar resistance to a wide range of antibiotics. Whole genome sequencing allowed the identification of single nucleotide variations in transcriptional regulators of oxidative stress-response: yfhP in SeSCar and soxR in SeLCar, which could be responsible for the increased resistance by improving the response to carvacrol and preventing its accumulation inside the cell. This study demonstrates the emergence of S. Typhimurium-resistant mutants against carvacrol, which might pose a risk to food safety and should therefore be considered in the design of food preservation strategies, or of cleaning and disinfection treatments.
Elisa Gayán, Elise Geens, Daniel Berdejo, Diego García-Gonzalo, Rafael Pagán, Abram Aertsen, and Chris W. Michiels
Elsevier BV
Daniel Berdejo, Elisa Pagán, Diego García-Gonzalo, and Rafael Pagán
Elsevier BV
Natalia Merino, Daniel Berdejo, Roberta Bento, Hesham Salman, María Lanz, Filippo Maggi, Susana Sánchez-Gómez, Diego García-Gonzalo, and Rafael Pagán
Elsevier BV
Geany Targino de Souza Pedrosa, Rayssa Julliane de Carvalho, Daniel Berdejo, Evandro Leite de Souza, Rafael Pagán, and Marciane Magnani
Wiley
AbstractThis study assessed the compromised acceptance threshold (CAT) and rejection threshold (RT) of Citrus lemon (CLEO) and Citrus reticulata essential oil (CREO) in apple and orange juices. The efficacy of CLEO and CREO concentrations below the RT were evaluated alone and combined with mild heat treatment (MHT) (54 °C, up to 12 min) to inactivate the autochthonous spoilage bacteria Lactobacillus brevis, Lactobacillus plantarum, and Leuconostoc mesenteroides in apple and orange juices. The CAT of CLEO and CREO varied from 0.15 to 0.17 μL/mL in orange and apple juices. The RT of CLEO was approximately 0.58 μL/mL in apple and orange juices, and the RT of CREO was 0.68 μL/mL in both juices. When CLEO and CREO were assayed alone, the highest concentration (0.50 μL/mL) decreased counts of all strains approximately 2 log10 CFU/mL after 12 min of exposure to 54 °C. All concentrations of CLEO or CREO in combination with MHT acted synergistically against L. brevis, L. plantarum, and L. mesenteroides. Decreases in counts varied with the strain, CLEO and CREO concentrations, juice type, and exposure time to the combined treatment. CREO was more effective than CLEO in combination with MHT against the strains in apple and orange juices. Effective combinations of CLEO or CREO with MHT to control the autochthonous spoilage bacteria did not compromise the quality parameters (°Brix, pH, and titratable acidity) that characterize unsweetened juices. These results indicate CLEO or CREO at concentrations below the sensory RT in combination with MHT as a feasible technology to control autochthonous spoilage bacteria in fresh fruit juices.Practical ApplicationThe present study provides novel information concerning the efficacy of sensorially accepted doses of CLEO and CREO combined with MHT against autochthonous spoilers in fruit juice. The valuable synergistic effects that can be observed when combining CLEO and CREO with MHT reveal a feasible preservation technology and alternative to traditional treatments that are successful because they help reduce treatment intensity, thereby avoiding adverse effects on the sensory, physicochemical, and nutritional properties of these products.
Daniel Berdejo, Beatriz Chueca, Elisa Pagán, Adriana Renzoni, William Kelley, Rafael Pagán, and Diego Garcia-Gonzalo
MDPI AG
Increased bacterial resistance to food preservation technologies represents a risk for food safety and shelf-life. The use of natural antimicrobials, such as essential oils (EOs) and their individual constituents (ICs), has been proposed to avoid the generation of antimicrobial resistance. However, prolonged application of ICs might conceivably lead to the emergence of resistant strains. Hence, this study was aimed toward applying sub-inhibitory doses of the ICs carvacrol, citral, and (+)-limonene oxide to Staphylococcus aureus USA300, in order to evaluate the emergence of resistant strains and to identify the genetic modifications responsible for their increased resistance. Three stable-resistant strains, CAR (from cultures with carvacrol), CIT (from cultures with citral), and OXLIM (from cultures with (+)-limonene oxide) were isolated, showing an increased resistance against the ICs and a higher tolerance to lethal treatments by ICs or heat. Whole-genome sequencing revealed in CAR a large deletion in a region that contained genes encoding transcriptional regulators and metabolic enzymes. CIT showed a single missense mutation in aroC (N187K), which encodes for chorismate synthase; and in OXLIM a missense mutation was detected in rpoB (A862V), which encodes for RNA polymerase subunit beta. This study provides a first detailed insight into the mechanisms of action and S. aureus resistance arising from exposure to carvacrol, citral, and (+)-limonene oxide.
Beatriz Chueca, Adriana Renzoni, Daniel Berdejo, Rafael Pagán, William L. Kelley, and Diego García-Gonzalo
American Society for Microbiology
ABSTRACT Food preservation by the use of essential oils (EOs) is being extensively studied because of the antimicrobial properties of their individual constituents (ICs). Three resistant mutants (termed CAR, CIT, and LIM) of Escherichia coli MG1655 were selected by subculturing with the ICs carvacrol, citral, and (+)-limonene oxide, respectively. These derivative strains showed increased MIC values of ICs and concomitantly enhanced resistance to various antibiotics (ampicillin, trimethoprim, chloramphenicol, tetracycline, kanamycin, novobiocin, norfloxacin, cephalexin, and nalidixic acid) compared to those for the parental strain (wild type [WT]). Whole-genome sequencing (WGS) of these hyperresistant strains permitted the identification of single nucleotide polymorphisms (SNPs) and deletions in comparison to the WT. In order to analyze the contribution of these mutations to the increased antimicrobial resistance detected in hyperresistant strains, derivative strains were constructed by allelic reversion. A role of the SoxR D137Y missense mutation in CAR was confirmed by growth in the presence of some ICs and antibiotics and by its tolerance to ICs but not to lethal heat treatments. In CIT, increased resistance relied on contributions by several detected SNPs, resulting in a frameshift in MarR and an in-frame GyrB ΔG157 mutation. Finally, both the insertion resulting in an AcrR frameshift and large chromosomal deletions found in LIM were correlated with the hyperresistant phenotype of this strain. The nature of the obtained mutants suggests intriguing links to cellular defense mechanisms previously implicated in antibiotic resistance. IMPORTANCE The antimicrobial efficacy of ICs has been proven over the years, together with their potential to improve traditional heat treatments by reducing treatment intensity and, consequently, adverse effects on food quality. However, the mechanisms of bacterial inactivation by ICs are still not well understood, in contrast to antibiotics. We performed WGS of three E. coli strains that are hyperresistant to ICs. The information provided detailed insight into the mechanisms of bacterial resistance arising from exposure to carvacrol, citral, and (+)-limonene oxide. Future experiments will undoubtedly yield additional insights into genes and pathways contributing to the acquisition of endogenous resistance to ICs.
E. Pagán, D. Berdejo, L. Espina, D. García-Gonzalo, and R. Pagán
Oxford University Press (OUP)
The application of essential oils in form of nanoemulsions has been proposed as a method to improve their solubility in aqueous solutions, and hence their antimicrobial activity. The objective of this study was to evaluate the antimicrobial activity of citral, applied directly or in combined treatments with heat or pulsed electric fields (PEF), as a function of the inoculation procedure assayed: (i) a simple, vigorous shaking method by vortex agitation (suspension of citral; s‐citral) or (ii) the previous preparation of nanoemulsions by the emulsion phase inversion (EPI) method (nanoemulsion of citral; n‐citral). n‐Citral was more effective in either inhibiting or inactivating Escherichia coli O157:H7 Sakai than s‐citral. However, when combined with heat, a greater synergistic effect was observed with s‐citral rather than with n‐citral, either in lab media (pH 7·0 and 4·0) or apple juice. For instance, while almost 5 log10 cell cycles were inactivated in apple juice after 15 min at 53°C in the presence of 0·1 μl ml−1 of s‐citral, the use of n‐citral required 30 min. The use of nanoemulsions did not modify the slight synergism observed when citral and mild PEF were combined (150 μs, 30 kV cm−1).
Laura Espina, Daniel Berdejo, Patricia Alfonso, Diego García-Gonzalo, and Rafael Pagán
Elsevier BV
Beatriz Chueca, Daniel Berdejo, Nelson J. Gomes-Neto, Rafael Pagán, and Diego García-Gonzalo
Frontiers Media SA
The improvement of food preservation by using essential oils (EOs) and their individual constituents (ICs) is attracting enormous interest worldwide. Until now, researchers considered that treatments with such antimicrobial compounds did not induce bacterial resistance via a phenotypic (i.e., transient) response. Nevertheless, the emergence of genotypic (i.e., stable) resistance after treatment with these compounds had not been previously tested. Our results confirm that growth of Escherichia coli MG1655 in presence of sub-inhibitory concentrations of the ICs carvacrol, citral, and (+)-limonene oxide do not increase resistance to further treatments with either the same IC (direct resistance) or with other preservation treatments (cross-resistance) such as heat or pulsed electric fields (PEF). Bacterial mutation frequency was likewise lower when those IC's were applied; however, after 10 days of re-culturing cells in presence of sub-inhibitory concentrations of the ICs, we were able to isolate several derivative strains (i.e., mutants) displaying an increased minimum inhibitory concentration to those ICs. Furthermore, when compared to the wild type (WT) strain, they also displayed direct resistance and cross-resistance. Derivative strains selected with carvacrol and citral also displayed morphological changes involving filamentation along with cell counts at late-stationary growth phase that were lower than the WT strain. In addition, co-cultures of each derivative strain with the WT strain resulted in a predominance of the original strain in absence of ICs, indicating that mutants would not out-compete WT cells under optimal growth conditions. Nevertheless, growth in the presence of ICs facilitated the selection of these resistant mutants. Thus, as a result, subsequent food preservation treatments of these bacterial cultures might be less effective than expected for WT cultures. In conclusion, this study recommends that treatment with ICs at sub-inhibitory concentrations should be generally avoided, since it could favor the emergence of hyper-resistant strains. To ascertain the true value of EOs and their ICs in the field of food preservation, further research thus needs to be conducted on the induction of increased transient and stable bacterial resistance via such antimicrobial compounds, as revealed in this study.