Alicia Rodríguez

Effect of climate change on Aspergillus flavus and aflatoxin B1 production

This review considers the available information on the potential impact of key environmental factors and their interactions on the molecular ecology, growth and aflatoxin production by Aspergillus flavus in vitro and in maize grain. The recent studies which have been carried out to examine the impact of water activity × temperature on aflatoxin biosynthesis and phenotypic aflatoxin production are examined. These have shown that there is a direct relationship between the relative expression of key regulatory and structural genes under different environmental conditions which correlate directly with aflatoxin B1 production. A model has been developed to integrate the relative expression of 10 biosynthetic genes in the pathway, growth and aflatoxin B1 (AFB1) production which was validated under elevated temperature and water stress conditions. The effect of interacting conditions of aw × temperature × elevated CO2 (2 × and 3 × existing levels) are detailed for the first time. This suggests that while such interacting environmental conditions have little effect on growth they do have a significant impact on aflatoxin biosynthetic gene expression (structural aflD and regulatory aflR genes) and can significantly stimulate the production of AFB1. While the individual factors alone have an impact, it is the combined effect of these three abiotic factors which have an impact on mycotoxin production. This approach provides data which is necessary to help predict the real impacts of climate change on mycotoxigenic fungi.

Quantification of ochratoxin A-producing molds in food products by SYBR Green and TaqMan real-time PCR methods

Ochratoxin A (OTA) is a mycotoxin synthesized by a variety of different fungi, most of them from the genera Penicillium and Aspergillus. Early detection and quantification of OTA producing species is crucial to improve food safety. In the present work, two protocols of real-time qPCR based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the non-ribosomal peptide synthetase (otanpsPN) gene involved in OTA biosynthesis. Seventy five mold strains representing OTA producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for OTA production by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). The ability of the optimized qPCR protocols to quantify OTA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1x10(4) to 10conidia/g per reaction for all qPCR assays in the different food matrices (cooked and cured products and fruits). The detection limit in all inoculated foods ranged between 1 and 10conidia/g for SYBR Green assay and TaqMan. No significant differences were found between the Ct values obtained from pure mold DNA and pure mold DNA mixed with food DNA. The ability of the designed qPCR methods to quantify two known conidial suspensions inoculated on several foods was evaluated. The amount of conidia assessed by both qPCR methods was close to the inoculated amount for most foods and indicates that the described procedure holds potential for use for the detection and quantification of OTA producing molds in foods.

TRI12 based quantitative real-time PCR assays reveal the distribution of trichothecene genotypes of F. graminearum and F. culmorum isolates in Danish small grain cereals.

Quantitative real-time PCR assays, based on polymorphisms in the TRI12 gene of the trichothecene pathway, were developed to identify and quantify the trichothecene genotypes producing 3-acetyl-deoxynivalenol (3ADON), 15-acetyl-deoxynivalenol (15ADON) or nivalenol (NIV) in the Fusarium graminearum species complex, Fusarium culmorum, Fusarium cerealis and Fusarium pseudograminearum. These assays were applied on a total of 378 field samples of cereal grain of wheat, barley, triticale, rye and oats collected from 2003 to 2007 to study the trichothecene genotype composition in Danish cereals. The three genotypes, 3ADON, 15ADON and NIV were found in all five cereal species, great annual variation in the occurrence of the trichothecene genotypes was evident with considerable variation between the samples. 3ADON was the dominant genotype in barley, triticale, rye and oats while 15ADON was most dominant in wheat. The NIV genotype was found at low levels in most samples. Study of genotype composition within the Danish F. graminearum and F. culmorum population was based on principal component analysis (PCA). PCA revealed that the dominating genotype of F. graminearum in wheat is 15ADON. For barley, the PCA analysis indicated that the F. graminearum population consisted of all three genotypes, and in triticale, the F. graminearum population consisted mainly of 15ADON genotype. F. culmorum/F. cerealis showed correlation to the NIV genotype in wheat and triticale but not in barley. F. culmorum/F. cerealis also showed some correlation to 3ADON especially in wheat and triticale. Selected wheat and barley samples from 1957 to 2000 showed low amounts of F. graminearum and F. culmorum in general but with a dominance of the 3ADON genotype. 15ADON was not detected in these samples, except for very low amounts in the sample representing the years from 1997 to 2000. Detection of low amounts of the 15ADON genotype in these historical samples and the relatively high amounts of 15ADON genotype in 2003 and following years correspond well with the occurrence of F. graminearum and indicates that the 15ADON genotype was introduced along with F. graminearum around 2000. The amounts of the 3ADON and 15ADON genotypes correlated well with the total amount of DON whereas the amounts of NIV genotype correlated well with the amount of NIV in wheat and triticale but not in barley where the results indicate that Fusarium poae may also contribute to the NIV content.

Presence of ochratoxin A on the surface of dry-cured Iberian ham after initial fungal growth in the drying stage

Accumulation of ochratoxin A (OTA) on the surface and to a 0.5 cm depth of dry-cured Iberian ham after initial fungal growth was investigated. For this, 20 dry-cured Iberian hams from the drying stage showing incipient fungal growth on the surface were analyzed. In addition, the presence of OTA-producing molds was examined on the surface of the hams by real-time quantitative PCR (qPCR) based on the otanpsPN gene. Quantification of specific OTA-producing molds, such as Penicillium nordicum and Penicillium verrucosum was also achieved on the hams by specific qPCR methods. Ten of 20 dry-cured hams showed OTA at higher levels than those established by legal regulation. OTA was even detected in the deep section of hams. OTA-producing molds ranged from 1.5 to 7.3 log cfu/cm². Accumulation of OTA on the hams seems to be related to the presence of OTA-producing molds and especially to P. nordicum.

The influence of salt (NaCl) on ochratoxin A biosynthetic genes, growth and ochratoxin A production by three strains of Penicillium nordicum on a dry-cured ham-based medium.

Iberian dry-cured ham is colonised by moulds during the ripening process. The environmental conditions occurring during the process including the salt content predisposes the surface to colonisation by Penicillium species, including Penicillium nordicum which can contaminate the curing ham with ochratoxin A (OTA). The objective of this study was to examine the effect of NaCl (10% and 22%=0.94 and 0.87 water activity (aw)) on the activation of two genes involved in the biosynthetic pathway for OTA production, otapksPN and otanpsPN, relative growth and phenotypic OTA production by three strains of P. nordicum (CBS 110.769, FHSCC1 and FHSCC2) on a ham-based medium over a period of 12days at 25°C. Growth of the three strains was faster at 0.87 than 0.94 aw on the ham-based media. However, some intra- and inter-strain differences were observed. Of the three strains, only two (CBS 110.789; FHSCC2) were able to express the two genes involved in the biosynthesis of OTA in the two salt treatments. RT-qPCR showed that the temporal expression of the two genes (otapksPN and otanpsPN) was relatively similar for the wild type strain (FHSCC2) at both 0.94 and 0.87 aw over the 12day period. However, in the type strain (CBS 110.769) expression increased rapidly at 0.94 aw but was significantly lower at 0.87 aw. Expression of these two genes occurred after 3day incubation, while phenotypic OTA production was observed only after 6days in the two toxigenic strains. The other strain did not produce any OTA. The OTA concentrations confirmed the results observed with the molecular tools. This suggests that the RT-qPCR gene expression of these two genes may be a good early indicator of potential contamination of dry-cured ham with OTA during dry-cured ham ripening.

Inhibition of ochratoxigenic moulds by Debaryomyces hansenii strains for biopreservation of dry-cured meat products.

The ability of the osmotolerant yeast Debaryomyces hansenii to inhibit Penicillium nordicum, the most common ochratoxigenic mould encountered in dry-cured meat products, was evaluated. The antagonistic effect of ten D. hansenii strains isolated from dry-cured ham was screened in vitro using malt extract media and meat extract peptone media with the water activity (a(w)) adjusted to 0.97 and 0.90. A significant inhibition of the two tested P. nordicum strains by D. hansenii cells and cell-free supernatants was observed. At 0.97 a(w), increasing D. hansenii inoculum concentrations significantly improved the inhibition of mould growth on solid medium, whereas at 0.90 a(w) this was not always the case. As observed by bright field microscopy, most D. hansenii strains were able to delay P. nordicum spore germination when co-cultured in malt extract broth. D. hansenii FHSCC 253H showed the highest overall in vitro inhibition of ochratoxigenic mould growth, and was therefore chosen for co-cultivation assays in dry-cured ham slices incubated at 0.94 and 0.84 a(w) simulating ham ripening. Regardless of the experimental conditions tested, lower levels of the inoculated P. nordicum strain were detected in co-cultivation batches compared with batches without D. hansenii. The highest level of mould growth inhibition was observed in batches at 0.94 a(w). Ochratoxin A (OTA) production in ham samples was detected by HPLC-MS. Co-culturing of P. nordicum with D. hansenii FHSCC 253H resulted in lower OTA levels compared with control samples without D. hansenii. The decrease of the mycotoxin presence due to D. hansenii FHSCC 253H was more efficient at 0.94 a(w) (OTA was below the detection limit). In conclusion, D. hansenii is potentially suitable as a biopreservative agent for preventing ochratoxigenic mould growth and OTA accumulation in dry-cured meat products. The inoculation of D. hansenii should be made at the beginning of processing (at the end of post salting) when the a(w) of the product is still high (near 0.94). This action in addition to application of appropriate hygienic actions and control of temperature and relative humidity throughout ripening is required to reduce health risks due to OTA exposure.

Climate change factors and Aspergillus flavus: effects on gene expression, growth and aflatoxin production

The objectives of this study were to obtain scientific data on the impact that interactions between water stress (water activity (aw); 0.97, 0.95, 0.92), temperature (34, 37 °C) and CO2 exposure (350, 650, 1000 ppm) may have on the growth, gene expression of biosynthetic genes (aflD, aflR), and phenotypic aflatoxin B1 (AFB1) production by a type strain of Aspergillus flavus on a conducive medium. The study showed that while aw affected growth there was no statistically significant effect of temperature or CO2 exposure. The effect of these interacting factors on aflD and aflR gene expression showed that at 34 °C there was maximum relative expression of aflD under the control conditions (34 °C, 350 ppm) with a decrease in expression with elevated CO2 and water stress. For aflR expression at 34 °C, there was a significant increase in expression, but only at 0.92 aw and 650 ppm CO2. However, at 37 °C, there was a significant increase in expression of both aflD and aflR at 0.95 and 0.92 aw and 650 and 1000 ppm...

Development of a multiplex real-time PCR to quantify aflatoxin, ochratoxin A and patulin producing molds in foods.

A multiplex real-time PCR (qPCR) method to quantify aflatoxin, ochratoxin A (OTA) and patulin producing molds in foods was developed. For this, the primer pairs F/R-omt, F/R-npstr and F/R-idhtrb and the TaqMan probes, OMTprobe, NPSprobe and IDHprobe targeting the omt-1, otanpsPN and idh genes involved in aflatoxin, OTA and patulin biosynthesis, respectively, were used. The functionality of the developed qPCR method was demonstrated by the high linear relationship of the standard curves constructed with the omt-1, otanpsPN and idh gene copies and threshold cycle (Ct) values for the respective producing molds tested to quantify aflatoxin, OTA and patulin producing molds. The ability of the optimized qPCR protocol to quantify producing molds was evaluated in different artificially inoculated foods (fruits, nuts, cereals and dry-ripened meat and cheese products). Efficiency values ranged from 81 to 110% in all inoculated foods. The detection limit was between 3 and 1logcfu/g for aflatoxin, OTA and patulin producing molds. The developed multiplex qPCR was shown be an appropriate tool for sensitive quantification of growth of toxigenic fungi in foods throughout the incubation time. Thus, the multiplex qPCR is a useful, rapid and efficient method to quantify simultaneously aflatoxin, OTA and patulin producing molds in food products.

Relationship between ecophysiological factors, growth and ochratoxin A contamination of dry-cured sausage based matrices.

Dry-cured sausages are colonised by moulds during the ripening process. The temperature and the salt content (which affects water activity, aw) predispose the surface to colonisation by Penicillium species, including Penicillium nordicum and Penicillium verrucosum which can lead to contamination of the sausages with ochratoxin A (OTA). The objective of this work was to obtain scientific data on the impact that interaction between ionic water stress (aw; 0.97, 0.94, 0.90, 0.87 and 0.84) and temperature (30, 25, 20, 15 and 10°C) may have on lag phases prior to growth, growth and OTA production by some P. verrucosum and P. nordicum strains isolated from dry-cured meat products on a dry-cured sausage-based medium over a period of 12days. Although P. nordicum had shorter lag phases than P. verrucosum, the latter grew faster than P. nordicum in most conditions tested. For both species, there was no growth and OTA production at 0.84 aw at all the temperatures tested. The fungi were more tolerant at moderate ionic aw conditions (0.94 and 0.90) and 20 and 25°C. In contrast, the patterns of production of OTA were very different from those for growth. Different OTA production profiles between the two OTA-producing species were found. While P. nordicum began producing OTA in most of the conditions tested by day 6, P. verrucosum only produced the toxin in these conditions when the temperature and aw were >10°C and >0.90, respectively. However, the P. verrucosum strain produced much higher concentrations of OTA than the P. nordicum strain in all conditions. We developed contour maps of the optimum and marginal aw×temperature conditions for growth/OTA production on dry-cured sausage-based medium for the first time. This suggests that these interacting conditions during the early phases of production must be effectively controlled as these favour growth of the toxigenic Penicillia. Knowledge on the ecophysiology of these two important Penicillium species on these matrices could help to make appropriate technological modifications during the sausage ripening process. Thus, our findings may help in informed decision-making in relation to temperature and salt additions at the beginning of processing/curing. Such changes may favour colonisation of starter cultures over OTA producing Penicillia and minimise OTA contamination risks in dry-cured sausages. This may be then effectively incorporated into the hygienic production system in the framework of HACCP.

Design of Primers and Probes for Quantitative Real-Time PCR Methods

Design of primers and probes is one of the most crucial factors affecting the success and quality of quantitative real-time PCR (qPCR) analyses, since an accurate and reliable quantification depends on using efficient primers and probes. Design of primers and probes should meet several criteria to find potential primers and probes for specific qPCR assays. The formation of primer-dimers and other non-specific products should be avoided or reduced. This factor is especially important when designing primers for SYBR(®) Green protocols but also in designing probes to ensure specificity of the developed qPCR protocol. To design primers and probes for qPCR, multiple software programs and websites are available being numerous of them free. These tools often consider the default requirements for primers and probes, although new research advances in primer and probe design should be progressively added to different algorithm programs. After a proper design, a precise validation of the primers and probes is necessary. Specific consideration should be taken into account when designing primers and probes for multiplex qPCR and reverse transcription qPCR (RT-qPCR). This chapter provides guidelines for the design of suitable primers and probes and their subsequent validation through the development of singlex qPCR, multiplex qPCR, and RT-qPCR protocols.