For anti-recombinant Z3276 protein polyclonal antibody preparation, Jiangsu Academy of Agricultural Sciences Institutional Animal Care and Use Committee approved the animal procedures (SYXK2015-0066) in the context of the guidelines of the Jiangsu Province Animal Regulations (Government Decree No. 45). New Zealand white rabbits were obtained from Jinling rabbit Farm (Nanjing, China), housed in cages (W 50 cm, L 30 cm, H 40 cm). They were provided with food and water ad libitum. At the end of the study, they were euthanized by intravenous injection of barbiturate, exsanguinated, and blood was clotted at 37◦C for 0.5 h, chilled at 4 ◦C for overnight. The purified recombinant protein Z3276 was used as antigen for polyclonal antibody preparation. Polyclonal antibody against Z3276 protein was obtained by immunizing New Zealand White rabbits subcutaneously at multiple sites with approximately 0.5 mg of purified protein emulsified 1:1 in Freund's complete adjuvant. The rabbits received one booster

injection with the same antigen concentration emulsified 1:1 with Freund's incomplete adjuvant 14 days later and then were bled 10 days after the booster was administered. Sera were stored in −80◦C freezer.

For anti-EHEC O157:H7 polyclonal antibody preparation, Jiangsu Academy of Agricultural Sciences Institutional Animal Care and Use Committee approved the animal procedures (SYXK2013-0014) in the context of the guidelines of the Jiangsu Province Animal Regulations (Government Decree No. 45). During feeding and study, health status of mice was monitored twice a day and recorded the clinical signs (ruffled hair coat, hunched posture, and diarrhea). If animals displayed clinical signs of illness, they were euthanized by cervical dislocation. Streptomycin-treated Balb/c mice were used to orally feed 3 × 108 CFU live EHEC O157:H7 strain 86-24 each week for 4 weeks. Mice were bled 2 weeks after the last booster. Sera were stored in a −80◦C freezer.

### Development Indirect ELISA for Detection Z3276 Expression in Vivo and in Vitro

For detection Z3276 expression in vivo, ELISAs were performed in 96-well plates (Costar, United States) coated with recombinant Z3276 antigen (0.6 µg/mL) and incubated at 4◦C overnight. Plates were washed and blocked with 1% bovine serum albumin (BSA) in phosphate buffered saline containing 0.05% Tween 20 (PBST). The mouse negative sera and anti-EHEC O157:H7 sera were, respectively, added to antigen-coated wells and incubated at 37◦C for 1 h. Sera were removed prior to adding goat antimouse IgG-HRP (1/5000 in PBST) (Boster, Wuhan, China) for 45 min at 37◦C. The substrate solution TMBS (Sigma) was added into the washed wells. After 5 min, 2 M H2SO4 was used to stop the ELISA followed by read absorbance at 450 nm.

For detecting Z3276 expression in vitro, the method was the same as above, with some differences: (1) For coating antigen preparation, the EHEC O157:H7 strain 86-24 was cultured in TSB medium for 6 h, and inactivated with 0.3% formaldehyde solution at 37◦C for 24 h until no live bacteria remained. The inactivated bacteria were washed two times with 0.01 M PBS (pH = 7.4) and used as antigen for coating plate; (2) the coating concentration was 108 CFU/mL; (3) The primary antibody was the recombinant Z3276 protein polyclonal antibody from rabbits.

### Construction of 1z3276 and the Complemented Strain

The mutant was generated using the suicide plasmid as described previously (Dozois et al., 2003). Briefly, DNA sequences flanking z3276 were amplified from the chromosomal DNA of EHEC O157:H7 strain EDL933 by PCR with two pairs of specific primers (P1-F/R and P2-F/R) carrying BamH I/EcoR I and Sal I/EcoR I restriction enzyme sites, respectively (**Table 2**). A DNA fragment containing the gentamicin resistance-encoding gene cassette was also obtained by PCR amplification from FastBac plasmid with primers (P3-F/R) carrying EcoR I restriction enzyme sites (**Table 1**). Gel-purified PCR products were cloned into pMEG375 to construct a suicide plasmid designated pMEG375 z3276FRGm, which was then transformed into E. coli strain CC118, named CC118-1. The positive plasmid pMEG375 z3276FRGm was transformed into a λpir lysogen of strain SM10, named SM10-1. The SM10-1 was hybridized with 86-24 strain on agar plate to produce 1z3276 mutant by homologous recombination. The 1z3276 was confirmed by PCR screening with z3276-specific primers (P4-F/R) (**Table 2**), DNA

#### TABLE 2 | Primers used in this study.

fmicb-09-01628 July 17, 2018 Time: 16:6 # 4

sequencing and Western blot analysis. The full-length z3276 gene was amplified by PCR (P5-F/R) from EDL933 genomic DNA to generate a complementation strain c1z3276. The PCR primers used are listed in **Table 2**. The amplified 1344 bp DNA fragment of the z3276 gene was inserted into the pMD19-T vector, and the recombinant plasmid was confirmed by DNA sequencing and restriction enzyme digestion. The positive pMD19-T-z3276 was introduced into the 1z3276 strain to overexpress the Z3276 protein for reconfirmation of its function.

### Crystal Violet Biofilm Assay

A static biofilm formation assay was performed in 96-well polystyrene plates as previously reported (Lourenço et al., 2013; Eberly et al., 2017). Briefly, overnight cultures were diluted to an OD600 of 0.05 in TSB medium and Minca medium, respectively (300 µL) and incubated for 10 h without shaking at 37◦C. After the cell density had been measured (turbidity at 630 nm), the plates were washed three times with PBS to remove all planktonic cells, and then 300 µL of 0.1% crystal violet was added to each well. After 20 min at room temperature, the microplate was emptied and washed three times with PBS. Next, 300 µL of 95% ethanol was added to resolve the stained biofilm cells. The total number of biofilm cells (absorbance at 570 nm) was measured and the total biofilm (OD570) was normalized with cell growth (OD630).

### Bacterial Adherence to Intestinal and Non-intestinal Cells

Cell adherence assays were performed as previously described with some modifications (Segura and Gottschalk, 2002). Bacteria were centrifuged, washed twice with PBS, and resuspended at 107 CFU/mL in RPMI 1640 medium without antibiotics. We employed human laryngeal carcinoma cell line HEp-2 cells and porcine neonatal jejunal epithelial cell line IPEC-J2 cells. Monolayers of these cells lines grown in 24-well plates were infected at a multiplicity of infection of 10 bacteria per cell. The plates were centrifuged at 800 × g for 10 min and incubated in RPMI 1640 medium with 2% newborn calf serum for 3 h at 37◦C with 5% CO2. After washing three times with PBS and digesting with a mixture of 0.25% tryptase and 0.02% EDTA, adherent bacteria were plated onto sorbitol MacConkey (SMAC) agar plates to count the CFU. All assays were performed in triplicate and repeated three times. The results were expressed as the adherence rate relative to that of the WT set as 100%.

### Adherence Inhibition Assays

Anti-Z3276 polyclonal antiserum, for which the sensitivity and specificity were demonstrated by western blot analysis, was made in our laboratory. Briefly, we constructed a pcold I-z3276 plasmid that could express abundant Z3276 protein in BL21 (DE3) cells. Purified Z3276 protein was used to immunize rabbits. Prior to adding onto the cell monolayer, 10 µL of the bacterial inoculum were incubated with 1:10, 1:50, and 1:100 dilutions of the anti-Z3276 and pre-immune bacterial suspensions at 37◦C with gentle agitation. The number of adherent bacteria per cell in each sample was determined by plating onto SMAC plates at 37◦C.

### Invasion Assay

The procedure for the invasion assay was the same as that for the adherence assay except the one ceftriaxone (10 µg/mL) and kanamycin (100 µg/mL) were added and incubated with the cells for 2 h to kill any extracellular bacteria before digestion. To confirm that all extracellular bacteria were killed by the antibiotic, 200 µL of the final PBS wash solution was plated on SMAC agar. Invasion frequencies were calculated as the number of bacteria surviving incubation with antibiotics divided by the total number of bacteria present in the absence of the antibiotic. All assays were performed in triplicate and repeated three times. The results were expressed as the invasion rate relative to that of the WT set as 100%.

### In Vivo Experiments

We used a streptomycin-treated mouse model to investigate the colonization ability of the strains. BALB/c mice were bought from experimental animal center of Yangzhou University (Yangzhou, China), housed in microisolator cages, provided with food and water ad libitum. To the study, the health status of mice was monitored twice a day and recorded the clinical signs (ruffled hair coat, hunched posture, and diarrhea). Thirty 6-week-old female BALB/c mice (n = 30) were divided into three groups and orally challenged, after anesthetized by inhaling diethyl ether, with WT, 1z3276, and c1z3276 strains at a dose of 1010 CFU in PBS, respectively. Mice infected with sterile PBS were used as controls. Fecal samples were taken on alternate days to monitor for shedding over 2 weeks.

### Twitching Motility Assay

Motility assays were performed as described previously with some modifications (Mattick, 2002). Briefly, 100 µL of an overnight culture was re-inoculated into 5 mL of sterile TSB and incubated at 37◦C without shaking until logarithmic phase. One microliter of each bacterial culture was dropped onto semisolid agar plates, which were incubated for 20 h at 37◦C before analysis. Motility was observed by measuring the diameter of the motility halo. Non-motile strains grew only at the site of inoculation.

### Statistical Analysis

fmicb-09-01628 July 17, 2018 Time: 16:6 # 5

Where appropriate, data were expressed as the mean ± SEM. The difference from two groups was analyzed using the Student'st-test and GraphPad Prism 5 software.

### RESULTS

### Phylogeny Analysis of Z2376 Protein

The BLAST analysis indicates that Z3276 amino-acid sequence has high identity between 91 and 100% to fimbrial proteins in GenBank (**Figure 1**), suggesting a conserved fimbrial domain in the Z3276 protein.

### Protein Expression and Purification

Recombinant plasmid pCold I-z3276 was sequenced to indicate that z3276 has 100% identity to reference sequences (GenBank CP015855.1) using Genscript Biotechnology, Co., Ltd. (Nanjing, China). Recombinant bacteria BL21/pCold I-z3276 was induced by IPTG. SDS-PAGE showed that recombinant Z3276 (33 kDa) was successfully expressed with 35% proportion to whole bacterial protein in contrast to naïve bacteria.

### Development of pAb Against Z3276 and EHEC O157:H7

Anti-Z3276 was successfully prepared from rabbits 229 and 230, and the titers were 2.6 × 106 and 7.9 × 105 , respectively, using indirect ELISA. Anti-EHEC O157:H7 was successfully prepared from 80% (4/5) mice, and the highest titer and the lowest titer were 9 × 104 and 3 × 103 , respectively.

### Indirect ELISA for Detection Z3276 Expression in Vivo and in Vitro

The ELISA plate coated with Z3276 was used to incubate with anti-EHEC O157:H7 sera and negative sera. The anti-EHEC O157:H7 sera gave an OD450 value greater than 1.0, compared with lower OD450 values from negative sera (**Table 3**). The detection data indicated Z3276 protein could express in vivo and perhaps on the bacterial surface.

The ELISA plate coated with the inactivated EHEC O157:H7 was used to incubation with anti-Z3276 sera and negative sera. The anti-EHEC O157:H7 sera gave OD450 value was greater than 1.0, compared with lower OD450 value from negative sera (**Table 4**). The detection data indicated Z3276 protein could express under the experimental conditions.

### Construction of a z3276-Defective Mutant

The 1z3276 mutant was constructed by homologous recombination and was confirmed by PCR (**Figure 2A**)

protein (yellow) encoded by the entire z3276 gene from EHEC O157:H7 genome. The scale bar represents 0.01 nucleotide substitutions per site.

#### TABLE 3 | Z3276 expression in vivo using indirect ELISA.

TABLE 4 | Z3276 expression in vitro using indirect ELISA.

and Western blot analysis (**Figure 2B**). PCR results confirmed that the 1z3276 mutant was negative for z3276 gene, and no reaction was detected between 1z3276 and anti-Z3276 serum by the Western blot assay. In term of growth stability, the 1z3276 and c1z3276 strains could be subcultured very well at least for 20 passages.

### Z3276 Contributes to Biofilm Formation

To test whether Z3276 was important for biofilm formation by EHEC O157:H7 on abiotic substrata, we compared the abilities of the WT, 1z3276, and c1z3276 strains to form biofilms in 96-well polystyrene plates. The results indicated that 1z3276 was reduced in its ability to form biofilms compared with the WT strain and complemented strains (**Figure 3**) regardless of culture media used. Mutant strains lost more abilities to form biofilms when cultured in TSB medium (P < 0.01) than in Minca medium (P < 0.05). Complemented strains regained stronger biofilm-forming abilities in a greater extent than WT strains had (P > 0.05).

### Anti-Z3276 Serum Blocked the Adherence of the WT Strain to Both HEp-2 and IPEC-J2 Cells

No significant difference was detected in the adherence ability of the 1z3276 mutant compared with the WT strain to both HEp-2 and IPEC-J2 cells (P > 0.05, P > 0.05, respectively) (**Figure 4A**). However, an obvious reduction was observed in the adherence of the WT strain to HEp-2 and IPEC-J2 cells after incubation with an anti-Z3276 antibody at a 1:2 dilution (antibody titer = 1:12,800) (P < 0.05, P < 0.05, respectively), and a 1:10 dilution (P < 0.05, P < 0.05, respectively) (**Figure 4B**), but not at a 1:100 dilution (P > 0.05, P > 0.05, respectively) (**Figure 4B**).

### Expression of Z3276 Could Trigger the Invasion of IPEC-J2 Cells

Invasion of host cells is a crucial step in bacterial pathogenesis. Compared with the WT strain, we found no change in the rate of invasion of HEp-2 cells for the 1z3276 mutant (P > 0.05), whereas an 81% reduction in invasion of IPEC-J2 cells was observed for the 1z3276 mutant (P < 0.05) (**Figure 5**). These results indicated that z3276 may be involved in the pathogenicity of EHEC O157:H7 in specific cell types.

### 1z3276 Mutant Showed Decreased Colonization in Mice

After treating mice with streptomycin (5 g/L for the first 3 days and then 0.5 g/L) and mitomycin C (2.5 mg/kg) to enhance their sensitivity (Zhang et al., 2011, 2014), we challenged with the WT and 1z3276 via oral–gastric inoculation. The results revealed shortened and lower fecal shedding for the 1z3276 compared with the WT strain (P < 0.05) (**Figure 6**), which indicated that Z3276 protein may be involved in colonization in vivo.

### Motility of the 86-24 WT and the 1z3276 Mutant Strains

Twitching motility is a phenomenon associated with virulence in many Gram-negative bacteria (Mattick, 2002), and is mediated by the retraction and extension of flexible pili by bacteria growing on a semi-solid surface. In this study, both the WT and 1z3276 were motile on agar. A 15-mm twitching motility zone on the semisolid agar surface was observed for the WT, compared with a 50% reduction in the zone for the 1z3276 mutant strain (P < 0.05) (**Figure 7**).

### DISCUSSION

EHEC is a zoonotic pathogen, of which O157:H7 is the most important serotype responsible for a number of outbreaks in

animals, poultry, and humans worldwide, leading to serious public health concern. The locus of enterocyte Effacement Island, a prophage producing two Shiga toxins and a 60 MDa virulence plasmid pO157 are considered the major determinants of EHEC O157:H7 pathogenesis (Sjogren et al., 1994; Schmedt et al., 1996). EspP was recently recognized as a new member of the serine protease autotransporters of Enterobacteriaceae family and, contributing to increased hemorrhaging into the intestinal tract (Khan et al., 2009) and evasion from immune system-mediated elimination (Orth et al., 2010). In addition to above-mentioned

FIGURE 5 | The ability of 1z3276 to invade IPEC-J2 cells was decreased compared with the wild-type strain. Ceftriaxone (10 µg/mL) and kanamycin (100 µg/mL) were added to ensure that only intracellular bacteria were obtained. The WT strain adhesion index was assumed to be 100%. The results shown are the means ± SEM of three independent experiments. ∗∗Indicates extreme significance at P < 0.01.

FIGURE 6 | 1z3276 showed shortened and lower fecal shedding in mice. Mice were orally challenged with strains WT, 1z3276, c1z3276, or PBS. Fecal samples were collected on alternate days over a 2-week period. The CFUs in each sample were determined by plating onto SMAC plates at 37◦C. The results shown are the averages of five mice per group.

defining virulence factors of EHEC O157:H7, there are other factors that may also contribute to its pathogenicity.

Increasing evidence suggests that fimbrial play an important role in the initial stages of EHEC O157:H7 infections (Xicohtencatl-Cortes et al., 2007). Martinez and coworkers

showed that the tip adhesin Fim H of type 1 fimbrial was sufficient to trigger invasion of uropathogenic E. coli into bladder epithelial cells (Martinez et al., 2000). However, the potential role of type 1 fimbrial in the pathogenicity of EHEC O157:H7 has not been reported, due to a nucleotide deletion and mutation in the fim operon encoding type 1 fimbrial (Li et al., 1997). Among the complete genome sequences available in current databases, the z3276 gene was only detected in EHEC O157:H7 (Li and Chen, 2012), and its amino acid sequence showed high homology with other E. coli type I fimbrial. Therefore, it is possible that z3276-encoding protein is a compensatory mechanism for type I fimbrial.

Biofilms help bacteria colonize inert surfaces, whilst protecting the bacterial cells from the host immune defense system as well as from antibiotic drugs (Gocer et al., 2017; Russell et al., 2018). In this study, an obvious reduction in biofilm formation was observed with a 1z3276 mutant compared with the WT strain in modified Minca medium and TSB, but no change was detected in Mueller–Hinton broth (MHB), brain heart infusion broth (BHI) or LB broth, suggesting that Z3276 production in vivo may be modulated by the composition of the medium and other conditions. Candida albicans biofilms are highly resistant to the actions of clinically important antifungal agents due to major multidrug efflux pumps encoded by C. albicans and the important role of biofilms in the drug resistance of planktonic cells (Cao et al., 2005). Moreover, E. coli biofilm bacteria showed weaker inflammatory responses and enhanced resistance to some antimicrobial peptides, and the increased in vivo survival of biofilm bacteria in a clinically relevant model of catheter infection has been reported (Lloyd et al., 2012; Chalabaev et al., 2014). Therefore, we can speculate that the z3276 gene in EHEC may confer the bacterium with resistance against antibiotics, thereby increasing its survival time in the host or the environment, which is critical to the transmission and infection.

Adherence is the first step in bacterial invasion. In the present study, we explored whether Z3276 plays a role in the adherence between the bacteria and cultured intestinal (IPEC-J2 cells) and non-intestinal (HEp-2 cells) epithelial cells. Qualitative analysis from IPEC-J2 cells infected with 1z3276 and WT showed no significant difference in the levels of adherence, with values of 1.2 × 105 CFU/mL and 1.6 × 105 CFU/mL, respectively. The same result was observed with HEp-2 cells. One possibility is that HEp-2 cells and IPEC-J2 cells do not possess a suitable receptor to interact with z3276-encoding protein, however, z3276-encoding protein may contribute to the adherence to other cell types or to

### REFERENCES

abiotic surfaces. For instance, the 1z3276 and its parental strain showed similar levels of invasion of HEp-2 cells. In contrast, the 1z3276 mutant showed 81% decrease in invasion ability to IPEC-J2 cells compared with the WT strain. Thus, we speculate that there might be certain moieties that can recognize Z3276 specifically and "carry" the bacterium into cells.

A mouse model was used to evaluate the colonization ability of the 1z3276 and the WT strains in vivo. The data of the animal study demonstrated that the 1z3276-infected mice rendered the animals lower bacterial counts and higher clearance efficiency, indicating that the ability of 1z3276 mutant to colonize the host and thereby survive in the host was impaired. This may be partially due to the reduced capacity to invade epithelial cells. The z3276 gene in EHEC may encode a fimbrial protein, or termed as tip adhesin (in the distal end of the pili), which has been previously reported to mediate the specific attachment to tissues or surfaces (Lindberg et al., 1987; Kaper et al., 2004; Low et al., 2006; Gu, 2017).

Besides as a colonization factor, Z3276 was also shown here to be involved in the twitching motility of EHEC O157:H7. This property may contribute to the pathogenicity of EHEC O157:H7, as it has been demonstrated that flagellum/fimbrial-mediated motility is essential for enhancing pathogen–host interactions and for promoting the subsequent adherence and colonization of several other Gram-negative pathogens (La Ragione et al., 2000; Wright et al., 2007; Mahajan et al., 2009).

In summary, this study confirmed that the z3276 gene in EHEC O157:H7 encodes multifunctional structures with properties that may contribute to host colonization and bacterial survival in the environment.

### AUTHOR CONTRIBUTIONS

XZ and KH conceived and designed the experiments. BZ and XS performed the experiments. HF analyzed the data. BZ and XZ wrote the paper.

### FUNDING

This work was supported by the National Natural Science Foundation of China (Grant No. 31572503), the Jiangsu Key Research and Development Plan (Grant No. BE2017341-1), and the National Key Research and Development Program of China (Grant No. 2018YFD0500500).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Zhang, Sun, Fan, He and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Combinational Inhibitory Action of *Hedychium spicatum* L. Essential Oil and γ-Radiation on Growth Rate and Mycotoxins Content of *Fusarium graminearum* in Maize: Response Surface Methodology

#### *Edited by:*

Pierina Visciano, Università di Teramo, Italy

#### *Reviewed by:*

Sunil D. Saroj, Symbiosis International University, India Vivek K. Bajpai, Dongguk University Seoul, South Korea Katarzyna Anna Marchwinska, ´ Poznan University of Economics, ´ Poland

#### *\*Correspondence:*

Naveen K. Kalagatur<EMAIL_ADDRESS>Venkataramana Mudili<EMAIL_ADDRESS>
#### *Specialty section:*

This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

*Received:* 14 September 2017 *Accepted:* 18 June 2018 *Published:* 31 July 2018

#### *Citation:*

Kalagatur NK, Kamasani JR, Siddaiah C, Gupta VK, Krishna K and Mudili V (2018) Combinational Inhibitory Action of Hedychium spicatum L. Essential Oil and γ -Radiation on Growth Rate and Mycotoxins Content of Fusarium graminearum in Maize: Response Surface Methodology. Front. Microbiol. 9:1511. doi: 10.3389/fmicb.2018.01511 Naveen K. Kalagatur 1 \*, Jalarama R. Kamasani 2 , Chandranayaka Siddaiah3 , Vijai K. Gupta4 , Kadirvelu Krishna5 and Venkataramana Mudili 5 \*

1 Food Microbiology Division, Defence Food Research Laboratory, Mysuru, India, 2 Freeze Drying and Processing Technology Division, Defence Food Research Laboratory, Mysuru, India, 3 Department of Biotechnology, University of Mysore, Mysuru, India, 4 Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia, 5 DRDO-BU-Centre for Life Sciences, Coimbatore, India

Nowadays, contamination of agricultural commodities with fungi and their mycotoxins is one of the most annoying with regard to food safety and pose serious health risk. Therefore, there is a requisite to propose suitable mitigation strategies to reduce the contamination of fungi and mycotoxins in agricultural commodities. In the present study, combinational inhibitory effect of Hedychium spicatum L. essential oil (HSEO) and radiation was established on growth rate, production of deoxynivalenol (DON) and zearalenone (ZEA) by Fusarium graminearum in maize grains. The HSEO was obtained from rhizomes by hydrodistillation technique and chemical composition was revealed by GC-MS analysis. A total of 48 compounds were identified and major compounds were 1,8-cineole (23.15%), linalool (12.82%), and β-pinene (10.06%). The discrete treatments of HSEO and radiation were effective in reducing the fungal growth rate and mycotoxins content, and the complete reduction was noticed at 3.15 mg/g of HSEO and 6 kGy of radiation. Response surface methodology (RSM) was applied to evaluate the combinational inhibitory effect of HSEO and radiation treatments on fungal growth rate and mycotoxins content. A total of 13 experiments were designed with distinct doses of HSEO and radiation by central composite design (CCD) of Stat-Ease Design-Expert software. In combinational approach, complete reductions of fungal growth, DON, and ZEA content were noticed at 1.89 mg/g of HSEO and 4.12 kGy of radiation treatments. The optimized design concluded that combinational treatments of HSEO and radiation were much more effective in reducing the fungal growth and mycotoxins content compared to their discrete treatments (p < 0.05). Responses of the design were assessed by second-order polynomial regression analysis and found that quadratic model was well fitted. The optimized design has larger F-value and adequate precision, smaller p-value, decent regression coefficients (R 2 ) and found statistically significant (p < 0.05). In addition, correlation matrix, normal plot residuals, Box-Cox, and actual vs. predicted plots were endorsed that optimized design was accurate and appropriate. The proposed combinational decontamination technique could be highly applicable in agriculture and food industry to safeguard the food and feed products from fungi and mycotoxins.

Keywords: mycotoxins, *Fusarium graminearum*, *Hedychium spicatum*, deoxynivalenol, zearalenone, essential oil, γ-radiation, response surface methodology

### INTRODUCTION

Mycotoxins are toxic secondary metabolites produced by filamentous fungi on agricultural products, which cause acute or chronic toxic effects in farm animals and humans called mycotoxicosis (Schirone et al., 2016; Du et al., 2017). The contamination of agricultural products with fungi occurs during pre-and post-harvesting stages due to inappropriate and unhygienic practices (Bernhoft et al., 2012). The Food and Agricultural Organization (FAO) of the United Nations estimate that ∼25% of agricultural products are contaminated with fungi and mycotoxins worldwide (Pitt and Hockings, 2009). The fungal infestation brings intolerable alterations in appearance, color, texture, flavor, and nutrition of food (Pitt and Hockings, 2009). Therefore, the incidence of fungi and mycotoxins contamination in agricultural products have become one of the foremost issues of farmers, food industry, and government concerning the food safety (Annunziata et al., 2017; Udomkun et al., 2017).

Among the mycotoxigenic fungi, Fusarium graminearum has received a wide attention because of its ability to produce a variety of mycotoxins, such as deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEA) under diverse climate conditions (Pasquali et al., 2016). Several studies have addressed the toxic effects of DON and ZEA in in-vitro and in-vivo experiments and documented their genotoxicity, hepatotoxicity, neurotoxicity, immunotoxicity, nephrotoxicity, reproductive, and developmental toxicity, carcinogenicity, etc. (Zinedine et al., 2007; Venkataramana et al., 2014; Schumann et al., 2016; Kalagatur et al., 2017; Gonçalves et al., 2018; Muthulakshmi et al., 2018; Reddy et al., 2018). Moreover, International Agency for Research on Cancer (IARC) has studied the carcinogenic property of DON and ZEA in laboratory animals and classified as the group 3 carcinogens (IARC, 1999).

The DON and ZEA have been detected in wide range of agricultural commodities, such as barley, corn, corn silage, hay, oats, rice, sesame seed, sorghum, and wheat (CAST, 2003; Zinedine et al., 2007). Particularly, Fusarium head blight (FHB) in wheat and barley, and Gibberella ear rot in maize caused by F. graminearum is a devastating plant disease of temperate regions and results in yield loss and mycotoxins contamination (Wilson et al., 2018). Most lately, Xu et al. (2018) from North China Plain have detected as high as 95.7% of maize germ contamination with DON and measured average attendance of DON in processed products of maize germs as 163.7– 1175.2 µg/kg. In Hungary, Tima et al. (2018) have surveyed the occurrence of DON in maize, wheat and its by-products during the period 2008–2015 and noticed overall mean of 2,159 ± 2,818 µg/kg, which was annoyingly much higher than maximum allowed limit. Besides, Mallmann et al. (2017) have summarized the occurrence of DON and ZEA in barley and wheat grains of Southern Brazil during the middle of 2008– 2015 and noticed 67% of DON and 41% of ZEA contamination. Similarly, Tralamazza et al. (2016) have observed 99% of DON and 84% of ZEA contamination in wheat samples originated from Brazil. Also, Ji et al. (2014) have detected 74.4% of wheat contaminated with DON in the FHB epidemic region of Jiangsu province, China. In our previous study (Mudili et al., 2014) 72–94 µg/kg of DON in freshly harvested maize grains from Andhra Pradesh, Karnataka, and Tamil Nadu states of Southern India was noticed. Furthermore, Mishra et al. (2013) have also measured the exposure and risk assessment of DON in the Indian individuals, and DON was identified in 30% of cereal samples and in that 7% of samples were surpassed the FSSR (Food Safety and Standard Regulation, India) limit of 1 mg/kg. In this context, F. graminearum and its toxins, DON, and ZEA have a great threat to the agricultural and food industry, especially in warm, and humid climate country like India (Ramana et al., 2011; Mishra et al., 2013; Divakara et al., 2014; Mudili et al., 2014; Aiyaz et al., 2016). In this scenario, there is a need to develop safe strategies to combat the problems of mycotoxigenic F. graminearum as well as DON and ZEA contamination in agricultural products.

Since last decade, agriculture and food industry have applied various decontamination practices to safeguard the agricultural products from fungi and mycotoxins (Zinedine et al., 2007; Kanapitsas et al., 2016; Kalagatur et al., 2018a,b). Unfortunately, some methods were undesirable, and only few of them were acceptable by WHO, FAO, EU, JECFA, and other regulatory agencies with some constraints. The application of synthetic fungicidal agents could drive the drug-resistant fungi, environmental pollution, health risk in animals and humans, and its use in food has been not passable and restricted worldwide (Kretschmer et al., 2009).  On the other hand, chemical decontamination methods bring unacceptable changes in the nutritional, sensory, and functional qualities of food and produce adverse toxic residues (Pitt and Hockings, 2009). The physical decontamination methods are most potent due to its spontaneous and strong effects. Particularly, radiation treatment is substantially efficient decontamination technique. The WHO and FDA of the United Nations have recognized that under well-established Good Manufacturing Practices (GMP) the application of γ-radiation in a low dose for disinfestations and enhancement of shelf life of agricultural commodities is nutritionally adequate and acceptable (Calado et al., 2014). Thus, a combination of biological and physical decontamination methods is likely to be a safe and much effective in controlling the fungal growth and mycotoxins content.

To the best of our knowledge, the combinational inhibitory action of essential oil and γ-radiation on the growth rate, production of DON and ZEA by F. graminearum has been not reported earlier. Therefore, the present study was aimed to establish the combinational inhibitory effect of Hedychium spicatum L. essential oil (HSEO) and radiation for the aforementioned purposes. The H. spicatum is a hardy and small perennial plant that belong to family Zingiberaceae and popularly known as "perfume ginger" or "spiked ginger lily" (Joshi et al., 2008). The HSEO is a rich source of diverse medicinal compounds, and it is widely used in herbal medicine to treat a variety of ailments, including nausea, stomachache, dysentery, local inflammations, asthma, bad breath, bronchitis, and rheumatic problems (Koul et al., 2005). The HSEO was obtained from rhizomes by hydrodistillation technique and its chemical profile was revealed by GC-MS analysis. The discrete inhibitory action of HSEO and radiation on the growth rate, production of DON, and ZEA by F. graminearum was evaluated in maize grains. Their combinational inhibitory action on aforementioned purposes was evaluated by response surface model adopting central composite design (CCD) and obtained model was validated experimentally.

### MATERIALS AND METHODS

### Chemicals and Reagents

Sabouraud dextrose agar (SDA), Sabouraud dextrose broth (SDB), and peptone were obtained from HiMedia (Mumbai, India). Certified standards of DON and ZEA were obtained from Sigma-Aldrich (Bengaluru, India). Immunoaffinity columns specific for DON and ZEA were procured from Vicam (Waters business, USA). The other chemicals used in the study were analytical grade and obtained from Merck Millipore (Bengaluru, India).

### Fungi Cultural Conditions

Fusarium graminearum (MTCC 1893) capable for the synthesis of DON and ZEA was obtained from Microbial Type Culture Collection and Gene Bank (MTCC), India, and grown on SDA for 7 days at 28◦C. The fungal spores were collected by soft scrape in sterile peptone solution supplemented with 0.01% Tween 80. The spore number was counted by hemocytometer and their number was set to 1 × 106 spores per mL.

### Collection and Characterization of *H. spicatum* L. Essential Oil

### Plant Material Collection and Essential Oil Extraction

The rhizomes of H. spicatum were obtained from agricultural field, Ooty, Tamil Nadu, India. Plants were dried under shade for 2 weeks at 27 ± 2 ◦C and ground to a fine powder (voucher no: PEO 37). The essential oil was extracted from 250 g of fine powder by hydrodistillation technique using Clevenger-type apparatus as per the procedure of European Pharmacopoeia (Council of Europe, 1997). The collected essential oil was dried over anhydrous sodium sulfate to get rid of moisture and safeguarded in an amber glass vial at 4◦C for further analysis.

### Chemical Characterization of HSEO by GC-MS

The chemical composition of HSEO was revealed by GC– MS analysis using PerkinElmer Clarus 600 C (PerkinElmer, Inc., Waltham, USA) equipped with DB-5MS column (30 m × 0.25 mm; 0.25µm film thickness) as per our previous reported methodology (Kalagatur et al., 2015). The chemical constituents of HSEO were identified by comparing their mass spectra (MS) with NIST/Wiley library and retention indices (RI) literature of Adams (2007). The quantification (%) of chemical constituents was obtained from the GC peak areas devoid of correction factors.

### Radiation Treatment

The radiation treatment was carried out in Gamma radiation chamber-5000 (Freeze Drying and Processing Technology Division, Defense Food Research Laboratory, Mysuru, India) and cobalt 60 was used as a source of γ-radiation with an effective dose frequency of 5.57 kGy per h at 35◦C. The absorbed radiation dose was determined by a Ceric-cerous dosimeter that was fixed to the surface of the bottom and top of the test sample. The equivalence of radiation dose was stated as Dmax/Dmin and it was 1.01 (Reddy et al., 2015).

### Assessment of Discrete and Combinational Inhibitory Effects of HSEO and Radiation Treatments on Growth Rate, Production of DON and ZEA by *F. graminearum* in Maize Experimental Design

The decisive aim of the present study was to evaluate the discrete and combinational inhibitory effects of HSEO and radiation treatments on the growth rate, production of DON and ZEA by F. graminearum in maize grains. The maize grains were obtained from the local agricultural market of Mysuru, Karnataka state, India, and 100 g was packed in plastic bags. The bags were autoclaved at 121◦C for 20 min and grains were dried out in a hot air oven at 60◦C. The autoclaving process used for removal of background microbial flora could affect the texture of the grains and which could have an impact on the results presented in this study.

The fungal spore suspension of 10 µL (1 × 106 spores per mL) was diluted to 1 mL with sterile peptone water solution and aseptically inoculated to the grain samples and mixed for 15 min at 140–160 rpm in a rotary shaker (Aziz et al., 2007). Following, grain samples were separately exposed to different doses of HSEO and radiation treatments to evaluate their discrete inhibitory effect. The HSEO was directly applied to maize grains and thoroughly vortexed and mixed for 15 min at 140–160 rpm in a rotary shaker as per methodology of Velluti et al. (2003). The combinational exposure of HSEO and radiation treatments with distinctive doses was accomplished by CCD of the response surface methodology (RSM) statistical program adopting State–Ease Design-Expert version <IP_ADDRESS> software application (Anderson and Whitcomb, 2016). In this study, grain samples initially treated with a distinctive concentration of HSEO were exposed to different doses of radiation treatment as per CCD. The grain samples were incubated for 14 days at water activity of 0.70 and temperature of 28◦C. The sample unexposed with HSEO and radiation treatments were referred as a control. Following the incubation period, the growth of fungi, quantification of DON, and ZEA were determined by colony forming units (CFU) and UHPLC, respectively.

The combinational inhibitory effect of HSEO and radiation (independent factors) on the reductions of CFU, DON, and ZEA content (response factors) was assessed by polynomial regression analysis of RSM. The lower and higher range of HSEO (0–3.15 mg/g) and radiation treatments (0–6 kGy) was fixed based on the dose required for complete inhibition of fungal growth and mycotoxins as per the study of their discrete exposure. The coded factors, name, units, range, levels, mean, and standard deviation of independent variables were shown in Supplementary Table 1. The order of experiments within the block was randomized and executed independently for six times. The regression analysis of attained responses i.e. log CFU, DON, and ZEA were assessed by fitting with suitable models represented by the second-order polynomial equation.

$$\mathbf{Y} = \beta\_0 + \sum\_{i=1}^{n} \beta\_{\mathbf{i}} \mathbf{x}\_{\mathbf{i}} + \sum\_{i=1}^{n} \beta\_{\mathbf{i}i} \mathbf{x}\_{\mathbf{i}}^2 + \sum\_{\mathbf{i} \neq \mathbf{j}=1}^{n} \beta\_{\mathbf{i}i} \mathbf{x}\_{\mathbf{i}} \mathbf{x}\_{\mathbf{i}\mathbf{j}}$$

where, "0" was the value of the fitted response at the center point of the design; i, ii, and ij were the linear, quadratic and crossproduct (interaction effect) regression terms, respectively and "n" denoted the number of independent variables.

### Determination of Fungal Growth by Dilution Plating Technique

The fungal growth in maize grains was determined as per methodology of Aziz et al. (2007) with minor modifications. Following the incubation period, 10 g of maize grains were collected from the plastic bags and suspended in 90 mL of sterile peptone water and mixed for 15 min at 140 – 160 rpm in a rotary shaker. The decimal dilutions were prepared in sterile peptone water and spread plated on SDA plates and incubated at 28◦C for 3 days. The fungal growth was determined in colony forming units (CFU) and results were expressed in log CFU/g.

### Quantification of DON and ZEA by UHPLC

Following the incubation period, 25 g maize grains were collected from the test samples and ground to a fine powder under aseptic conditions and suspended in 250 mL solution of acetonitrile and water (v/v, 6:4). The blend was mixed for 15 min at 140– 160 rpm and the supernatant was collected by centrifugation at 6000 rpm for 5 min. The supernatant was subjected to clean-up with immunoaffinity columns of DON and ZEA in according to the instructions of the manufacturer (Vicam, Waters business, USA). The quantification DON and ZEA were done as per our previous reported methodology (Mudili et al., 2014; Kalagatur et al., 2015) using UHPLC system (Nexera, Shimadzu, Japan). The quantification of DON and ZEA was deducted from their respective standard calibration curve and concentration was expressed in µg/g.

### Statistical Analysis

The experiments were performed in six independent replicates. The statistical analysis was done according to the one-way ANOVA and significant differences were determined by Tukey's post hoc multiple comparison test and value of p < 0.05 was considered significant. The statistical analysis and graphical illustrations were attained adopting the software program GraphPad Prism trial version 7 (GraphPad Software, Inc., USA). The statistical analysis for the optimization of RSM was done by CCD following the State–Ease Design-Expert trial version 10 software program (Stat-Ease, Inc., Minneapolis, USA). The responses of the polynomial regression at a significance level of p < 0.05 were considered to design statistical model (Anderson and Whitcomb, 2016). The accuracy of the model was evaluated by measuring the coefficient of determination (R 2 ) and lack of fit.

### RESULTS AND DISCUSSION

### Chemical Composition of HSEO

In the present study, the chemical composition of HSEO was revealed by GC-MS and a total of 48 compounds were identified constituting to 96.84% of total weight (**Table 1**). The major compounds were 1,8-cineole (23.15%), linalool (12.82%), βpinene (10.06%), γ-terpinene (8.16%), terpinolene (5.04%), αterpinene (3.81%), and α-terpineol (3.35%). In our study, βeudesmol, furanoid, β-himachalene, hedycaryol, eremoligenol, agarospirol, 8-epi-β-bisabolol, and α-cadinol were not detected in accordance with the previous reports of Joshi et al. (2008) and Sabulal et al. (2007). While, diverse compounds, such as terpinolene, α-terpinyl acetate, γ-eudesmol, and γ-muurolene were identified in our study. The chemical compounds and their concentration of essential oil depend on the genetics of plant, part of the plant used, geographical origin, nutrients, harvesting time, and analytical method employed (Gobbo-Neto and Lopes, 2007). Therefore, in our study chemical compounds and their concentration values were varied in comparison to the previous reports.

### Inhibitory Effect of HSEO and Radiation Treatments on Growth Rate, Production of DON and ZEA by *F. graminearum* in Maize Discrete Treatment of HSEO and Radiation

In the present study, discrete treatment of HSEO and radiation were effective in reducing the fungal growth (log CFU), production of DON and ZEA by F. graminearum in maize grains. A quantity of 5.79 ± 0.33 of log CFU/g, 6.24 ± 0.37 of DON (µg/g), and 8.67 ± 0.45 of ZEA (µg/g) were determined in the control sample. While, log CFU, DON, and ZEA content were reduced in test samples in a dose-dependent way with treatment of HSEO and radiation (**Tables 2**, **3**). The fungal growth, DON, and ZEA content were not detected at 3.15 mg/g TABLE 1 | Chemical composition of Hedychium spicatum L. essential oil determined by GC-MS analysis.

\*Actual retention indices of compounds on DB-5 column. #Retention indices of compounds on DB-5 column in accordance to literature of Adams (2007).

TABLE 2 | Discrete inhibitory effect of H. spicatum essential oil (HSEO) on growth rate (log CFU), production of deoxynivalenol (DON) and zearalenone (ZEA) by F. graminearum in maize grains.

The data was processed by one-way ANOVA following Tukey's test and the columns with same alphabetic letters were not significant (p < 0.05).

The data was processed by one-way ANOVA following Tukey's test and the columns with same alphabetic letters were not significant (p < 0.05).

of HSEO and 6 kGy of radiation. The linear regression curves for reductions of log CFU, DON, and ZEA content were constructed against different doses of HSEO and radiation (**Figures 1**, **2**). The obtained regression models exhibited the goodness of fit (R 2 ) close to 1 and found statistically significant (p < 0.05; **Tables 4**, **5**). The regression models confirmed that reductions of log CFU, DON, and ZEA content by HSEO and radiation were dose-dependent.

Best of our knowledge, till the date, the fungicidal activity of HSEO on F. graminearum was not reported, and this is the first report. On the other hand, Pawar and Thaker (2006) has determined the antifungal activity of HSEO on Aspergillus niger by a zone of inhibition assay as 8 mm. The growth inhibitory activity of radiation treatment on F. graminearum was less investigated. In support of our study, very few reports are available on the application of radiation treatment in the management of F. graminearum in food and feed matrices. Aziz et al. (2007) have reported the complete inhibition of Fusarium spp. growth at 4 kGy in barley and 6 kGy in wheat and maize grains. On the other hand, Ferreira-Castro et al. (2007) and Lima et al. (2011) have documented that high dose of 10 kGy radiation treatment was required for complete reduction of Fusarium spp. in maize and cowpea bean grains, respectively. The

FIGURE 1 | Linear regression curve for growth inhibitory activity of different doses of (A) H. spicatum L. essential oil (HSEO) and (B) radiation treatments on F. graminearum in maize. The log CFU/g was declined with the dose of HSEO and radiation. The data was processed by one-way ANOVA following Tukey's test and value of p < 0.05 was considered significant.

radiation dose required for reduction of fungal growth depends on certain factors, such as the type of species, population number, intensity of pigmentation, water content, genetics, and induction of protective enzymes, which play a major role in the regaining of the damage induced by radiation (Jeong et al., 2015). In the present study, the background microbial flora of maize grains was removed by autoclave sterilization and which could affect the texture of the grains and have an impact on the results. Therefore, the differences in the radiation dose required for complete reduction of fungal growth were noticed in present and previous studies.

### Combinational Treatment of HSEO and Radiation

Radiation process is measured as an ideal and beneficial decontamination technique at a low dose. High radiation dose produces intolerable features and toxic substances in food, such as structural and chemical changes, nutritional and sensory loss, undesirable odor and flavor, and rancidity (Calado et al., 2014). Therefore, minimally radiation processed foods are highly preferred and acceptable by consumer and regulatory bodies. In this context, food industry and food technologists have made many efforts through last decade to reduce the efficient radiation dosage rate and augment the decontamination efficiency of radiation. Currently, combining radiation with other decontamination agents, such as essential oils, chemical preservatives, and modified atmospheres is an effective and innovative way to improve the decontamination efficiency of radiation (Ghosh et al., 2017; Sirocchi et al., 2017; Wilson et al., 2017). The combinational approach reduces the possible means radiation dose required for decontamination of microbial flora and could promote food safety at low radiation dosage.

The combinational inhibitory effect of HSEO and radiation treatments on the fungal growth (log CFU), production of DON and ZEA by F. graminearum in maize grains were executed following the CCD of RSM statistical program (Wu et al., 2017). The RSM is a collection of statistical and mathematical techniques, and one of the widely used applications in the design, development, and invention of new process or products, and improvement of an existing process or product designs in food science and technology. The RSM requires only a minimal TABLE 4 | Linear regression curve for discrete inhibitory effect of H. spicatum essential oil (HSEO) on growth rate (log CFU), production of deoxynivalenol (DON) and zearalenone (ZEA) by F. graminearum in maize grains.

TABLE 5 | Linear regression curve for discrete inhibitory effect of radiation treatment on growth rate (log CFU), production of deoxynivalenol (DON) and zearalenone (ZEA) by F. graminearum in maize grains.

number of experiments between input variables that potentially influence performance measures or quality characteristics of the product or process and allow to identify breakthrough productive information by means of reducing cost, errors, and disturbance. The relations between the input factors and responses were hypothesized or assessed by choosing a model (Whitcomb and Anderson, 2004).

The actual or obtained response of 13 CCD experiments of the present study was provided in **Table 6**. The data was analyzed by second-order polynomial regression analysis employing the software Design Expert (version 10.0.6) to optimize the response surface models.

The correlation matrix of regression coefficients of independent factors was correlated with one another on a TABLE 6 | Central composite design (CCD) for combinational inhibitory action of H. spicatum essential oil (HSEO) and radiation treatments (independent factors) on the reductions of log CFU, DON, and ZEA (responses).

scale of perfect negative correlation (−1) to perfect positive correlation (+1) as per Pearson's correlation coefficients to conclude their appropriateness. The perfect negative correlated matrix was appropriate and agrees to the individual effect of independent factors on responses (Anderson and Whitcomb, 2016). In the present study, a perfect negative correlation matrix was observed for both independent factors (**Figure 3**). A perfect negative correlation matrix of − 0.680 and − 0.602 (log CFU), − 0.681 and − 0.625 (DON), and − 0.679 and − 0.632 (ZEA) were observed for HSEO and radiation treatments, respectively. It indicated that independent factors were independently effective in reducing the log CFU, DON, and ZEA. These results were in accordance with the assessment of the discrete inhibitory effect of HSEO and radiation treatments on the reductions of log CFU, DON, and ZEA content (**Figures 1**, **2**). Therefore, the combinational assessment of HSEO and radiation treatments for the aforementioned purposes were well appropriate and could reveal newer insights. Consequently, combinational treatments of HSEO and radiation were highly effective in reducing the log CFU, DON, and ZEA content in maize grains. The complete reductions of fungal growth, DON, and ZEA content were noticed at combination of 1.89 mg/g of HSEO and 4.12 kGy of radiation treatments (**Table 11**). The dose of HSEO (1.89 mg/g) and radiation (4.12 kGy) required for complete reductions of fungal growth, DON, and ZEA content were much less in combinational treatment compared to discrete treatments of HSEO (3.15 mg/g) and radiation (6 kGy).

The obtained CCD results of the RSM study concluded that quadratic model was well appropriate for all the responses (log CFU, DON, and ZEA). The coefficient of independent variables in terms actual factors for second-order polynomial equation designed for the responses attained as below:

$$\begin{aligned} \text{Log CFU/g} &= +6.87 - 2.98 \ast \text{HSEO} - 1.27 \ast \text{radiation} \\ &+ 0.26 \ast \text{HSEO} \ast \text{radiation} + .0.34 \ast \text{HSEO}^2 \\ &+ 0.05 \ast \text{radiation}^2 \end{aligned}$$

The significance of the second-order polynomial model was evaluated by analysis of variance (ANOVA) and coefficient of determination (R 2 ). The ANOVA results for the fitted quadratic polynomial models of log CFU, DON, and ZEA were presented in **Tables 7**, **8**, **9**, respectively. For any of the terms in the model, a greater F-value and smaller p-value would indicate a more significant effect on the respective response variables (Atkinson and Donev, 1992). The present model has larger Fvalue of 251.61 (log CFU), 335.88 (DON), and 229.45 (ZEA) and the associated p-value <0.0001 and implied that the optimized models were very significant. Furthermore, F-value and p-value of the lack of fit were 4.44 and 0.0918 for log CFU, 2.37 and 0.2120 for DON, 2.24 and 0.2255 for ZEA, respectively (**Table 10**). Which indicated that lack of fit was not significant and 9.18%, 21.20%, and 22.55% chance could occur due to noise in log CFU, DON, and ZEA, respectively. The goodness of fit of the models was judged by estimating the coefficient of determination (R 2 ). The value of R 2 for log CFU, DON, and ZEA were observed as 0.9945, 0.9958, and 0.9939, respectively. Which implies that 99.45%, 99.58%, and 99.39% of the variations could be explained by the fitted models of log CFU, DON, and ZEA, respectively. For a good statistical model, the predicted Rsquared value should be close to adjusted R-squared value and the obtained differences in all the responses of the present study were appropriate (**Table 10**). Also, adequate precision should be >4.0 is desirable for the significant model and adequate precision of 52.343 (log CFU), 60.519 (DON), and 50.190 (ZEA) in the

TABLE 8 | ANOVA for DON (µg/g) response surface quadratic model.

TABLE 9 | ANOVA for ZEA (µg/g) response surface quadratic model.

present study indicates an adequate signal and the obtained models were well appropriate to navigate the design space (**Table 10**).

The diagnostic and correlation plots were judged to endorse the obtained significance of ANOVA. The normal plot residuals of normal % probability vs. externally studentized residuals were constructed to illustrate the accuracy of optimized design (**Figure 4**). The distribution of externally studentized residuals was satisfactorily followed the normal distribution. Which indicated that residuals plots follow linear behavior and the predicted model is accurate (Myers et al., 2016). A Box-Cox plot for power transforms of responses were considered to determine the most appropriate power law transformation to fit the responses. The best recommended transform (λ) of 0.80, 0.94, and 0.91 were observed for log CFU, DON, and ZEA, respectively (**Figure 5**). The obtained λ values were close to the current value of 1 for none and which indicates that responses were well fitted by the optimized design (Box and Cox, 1964). The optimized designs were assessed to examine the correlation between the actual and predicted responses (**Figure 6**). The obtained data points were close to the straight line and presented a good correlation coefficient (R 2 ). The correlation curves suggested a high degree correlation of 0.9945, 0.9958, and 0.9939 between the actual and predicted values of the log CFU, DON, and ZEA, respectively and concluded that the fundamental assumptions of the analysis were well appropriate. The fitted polynomial equation was expressed as 3D surface plots to illustrate the interactive effects of the independent variables on the responses and to deduce optimum conditions (**Figure 7**). In conclusion, appropriateness of the optimized model for predicting the optimum responses was verified with suggested optimum conditions. The obtained actual values of suggested experiments were found in agreement with the predicted values (**Table 11**). The complete reductions of fungal growth (log CFU), DON, and ZEA content were noticed in maize grains at combinational treatment of 1.89 mg/g of HSEO and 4.12 kGy of radiation. These results suggested that the designed quadratic models for reductions of log CFU, DON, and ZEA content in maize grains were statistically significant and could adequately represent the real interdependence of factors chosen (HSEO and radiation).

The present study concluded that combinational exposure of HSEO and radiation on the reductions of fungal growth, DON, and ZEA content by F. graminearum in maize grains was highly efficient compared to their discrete exposure. In combinational approach, radiation dose required for complete reductions of fungi and mycotoxins were reduced from 6 kGy (discrete treatment) to 4.12 kGy. Which indicated that radiation treatment with the combination of HSEO was highly efficient and minimally radiation processed compared to unaccompanied treatment of radiation. However, structural, chemical and nutritional changes of maize grains were not determined in the present study. In support of our study, earlier reports demonstrated that essential oils with a combination of modified atmosphere packaging, mild heat treatment, and radiation were highly effective and safe in maintaining the microbiological safety of the food (Hossain et al., 2014). Several studies noticed that antifungal activity of essential oils is reliant on chemical constituents and their synergistic action (Hyldgaard et al., 2012; da Cruz Cabral et al., 2013). In our study, varieties of chemical compounds were noticed in HSEO (**Table 1**) and the attributed antifungal activity of HSEO against F. graminearum could be due to individual and combination of action of 1,8-cineole, linalool, β-pinene, limonene, germacrene-D, γ-terpinene, terpinolene, αterpinene, and α-terpineol (Velluti et al., 2003; Van Vuuren and Viljoen, 2007; Deba et al., 2008; Chee et al., 2009; Vilela et al., 2009; Gao et al., 2016). These compounds could alter the integrity of cell membrane and disturb the membrane fluidity, osmotic balance, enzymatic functions, and leak cytoplasmic contents of the cell. These cascade of events stops the ATP synthesis, growth and proliferation of fungi, and activates the caspase-mediated apoptotic death by releasing the mitochondrial cytochrome c and death receptors (Ramsdale, 2008; Usta et al., 2009; Tian et al., 2012; Chen et al., 2014). In case of radiation treatment, radiolysis of cellular water of organisms produces positive-charged water radicals (H2O+) and negative-charged free electrons (e−), and a series of cross-combination and recombination reactions generate highly reactive molecules (de Campos et al., 2004; Le Caër, 2011). These molecules attack and cleave hydrogen from sugar and bases of nuclear material, and destruct the other cellular components, i.e., protein and lipids and even damage the integrity of cellular membranes. This process inhibits spore germination and biomass production and promotes the death of fungi (Calado et al., 2014).

Henceforth, combined treatments of HSEO and radiation presented a superior antifungal efficiency on F. graminearum than the discrete treatment of HSEO or radiation alone. Moreover, many reports showed that the combinational treatment of essential oils with radiation up to a dose of 10 kGy do not produce any significant changes in the quality and quantity of essential oils and not produce any toxic residues (Chatterjee et al., 2000; Haddad et al., 2007). Consequently, functional features of essential oils do not effect on exposure to radiation and acceptable by Institute of Food Science & Technology (IFST), WHO, FAO, International Atomic Energy Agency (IAEA), and consumer (Lacroix and Ouattara, 2000; IAEA, 2001; Maherani et al., 2016). Henceforth, the proposed novel combinational exposure of HSEO and radiation could be highly acceptable and efficient for reductions of fungal growth and mycotoxins in food and feed matrices and thereby support in

obtaining the highly microbiological safe food products. Though, a greater understanding of the combinational antifungal activity of essential oils and radiation is required at the cellular and molecular level to exploit underlying the molecular death machinery.

growth), production of (B) DON (µg/g) and (C) ZEA (µg/g) by F. graminearum in maize grains.

## CONCLUSION

In the present study, the discrete and combinational inhibitory effects of HSEO and radiation treatments on growth, production of DON and ZEA by F. graminearum in maize grains were assessed under laboratory set-ups. The GC-MS analysis of HSEO revealed the presence of 48 compounds constituting to 96.84% of total weight and major compounds were 1,8 cineole, linalool, and β-pinene. The simple linear regression analysis showed that HSEO and radiation treatments were discretely inhibited the fungal growth, DON, and ZEA content in maize grains by the dose-dependent way, and complete inhibition were noticed at 3.15 mg/g of HSEO and 6 kGy of radiation. The combinational inhibitory action of HSEO and

TABLE 11 | Assessment of proposed predicted values of design with actual values of experiment to maximize the inhibition of growth rate, DON, and ZEA by F. graminearum in maize grains.

radiation treatments on growth, production of DON and ZEA by F. graminearum in maize grains was studied by CCD of RSM statistical program. The fungal growth, DON, and ZEA content were not detected at combinational treatment of 1.89 mg/g of HSEO and 4.12 kGy of radiation. The results revealed that combination of HSEO and radiation treatments could reduce the fungal growth, DON, and ZEA content at much lower concentration than their discrete inhibitory dose. The quadratic model was found well appropriate for reductions of fungal growth (log CFU), DON, and ZEA content in maize grains. The optimized design was found statistically significant (p < 0.05) with larger F-value and adequate precision, and a smaller pvalue. The actual data points were close to the straight line and presented good correlation regression coefficients (R 2 ) for the responses. In addition, diagnostic and correlation plots, i.e., correlation matrix, normal plot residuals, Box-Cox, and actual vs. predicted plots were confirmed that optimized design was accurate and appropriate. The study concluded that optimized design could adequately represent the real interdependence of factors chosen (HSEO and radiation) for reductions of fungal growth, DON, and ZEA content. The present study suggests that combinational treatment of essential oil and radiation could be a novel promising approach for improving the shelflife and safety of food and feed matrices. As well, study accomplishes that usage of natural antifungal agents with the combination of food-processing techniques is an appropriate

### REFERENCES

and innovative strategy to improve decontamination efficiency of food-processing techniques.

### AUTHOR CONTRIBUTIONS

NK, JK, CS, KK, and VM designed and interpreted data of the work. NK, JK, CS, VG, and VM have drafted the work. All authors have approved the final version to be published.

### ACKNOWLEDGMENTS

The first author is thankful to the UGC, Government of India for providing Junior Research Fellowship [File no: F. 2-14/2012(SA-I)] to pursue Ph.D. Authors are also grateful to Dr. K. Jayathilakan and Dr. M.C. Pandey, Freeze Drying and Processing Technology Division, Defence Food Research Laboratory, Mysuru, India, for providing the γ–radiation facility. The authors are also thankful to Director, DFRL, and DRDO-BU-CLS for their kind support.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.01511/full#supplementary-material

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Kalagatur, Kamasani, Siddaiah, Gupta, Krishna and Mudili. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Cronobacter sakazakii and Microbiological Parameters in Dairy Formulas Associated With a Food Alert in Chile

Julio Parra-Flores1 \*, Fabiola Cerda-Leal1 , Alejandra Contreras1 , Nicole Valenzuela-Riffo1 , Alejandra Rodríguez1 and Juan Aguirre2

1 Molecular Microbiology Laboratory, Faculty of Health and Food Sciences, Universidad del Bío-Bío, Chillán, Chile, 2 Department of Agricultural Industry and Enology, Universidad de Chile, Santiago, Chile

#### Edited by:

Giovanna Suzzi, Università di Teramo, Italy

### Reviewed by:

Chiara Montanari, Università degli Studi di Bologna, Italy Antonio Martínez, Consejo Superior de Investigaciones Científicas (CSIC), Spain

#### \*Correspondence:

Julio Parra-Flores<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

Received: 16 April 2018 Accepted: 09 July 2018 Published: 31 July 2018

#### Citation:

Parra-Flores J, Cerda-Leal F, Contreras A, Valenzuela-Riffo N, Rodríguez A and Aguirre J (2018) Cronobacter sakazakii and Microbiological Parameters in Dairy Formulas Associated With a Food Alert in Chile. Front. Microbiol. 9:1708. doi: 10.3389/fmicb.2018.01708 The objective of this study was to evaluate the presence of Cronobacter sakazakii and microbiological parameters in dairy products associated with a food alert. Ninety dairy product samples were analyzed, including seven commercial brands and two product types (liquid and powdered) from four countries. Aerobic plate count (APC) and Enterobacteriaceae count were performed according to Chilean standards. Cronobacter spp. and C. sakazakii were identified by polymerase chain reaction real time amplification of rpoB and cgcA genes and the genotype by multilocus sequence typing. Eighty-eight percent of dairy products showed APC higher than the detection limit. Fifty percent of liquid commercial brand samples contained APC: 2.6, 2.3, 1.1, and 2.9 CFU/mL in brands A, C, E, and G, respectively. Results for powdered commercial brands were 3.0, 3.6, and 5.7 CFU/g in brands B, D, and F, respectively. Maximum count (5.7 CFU/g) occurred in brand F dairy product manufactured in Chile. Enterobacteriaceae were found in 55% of the samples, 64% in liquid and 51% in powdered commercial brands. In 50% of brands B, D, and E, samples contained 2.9, 2.8, and 2.7 log CFU/g, respectively. Only liquid commercial brands from the United States had Enterobacteriaceae values between 0.1 and 4.5 CFU/mL. Seventeen suspicious strains were isolated and nine were identified as Enterobacter spp. Only eight suspicious strains from four powdered commercial brands (Chile and Singapore) were confirmed as C. sakazakii by rpoB and cgcA gene amplification and fusA sequencing. C. sakazakii prevalence in the analyzed samples was 8.8%. There were 11% of powdered milk brands that contained APC between 4.0 and 4.7 log CFU/g and 55% of the samples contained Enterobacteriaceae. C. sakazakii was found in dairy products manufactured in Chile and Singapore. On the basis of this information, the Chilean Ministry of Health (RSA) decreed a national and international food alert and recalled all the product batches that resulted positive in the present study from supermarkets and pharmacies.

Keywords: Cronobacter sakazakii, food alert, microbiological parameters, powdered infant formula, liquid dairy formula

## INTRODUCTION

fmicb-09-01708 July 27, 2018 Time: 17:1 # 2

On June 2, 2017 the Chilean Ministry of Health issued a national and international food alert as a result of the presence of Cronobacter sakazakii in two powdered formula samples intended for children under 10. Researchers from the Universidad del Bío-Bío conducted a study which led to the food alert. This preventive measure was adopted because of the risk of disease associated with Cronobacter spp. and C. sakazakii in hypersensitive groups of the population (Food and Agriculture Organization and World Health Organization [FAO/WHO], 2008; Jason, 2015).

Cronobacter spp. was initially defined as the new bacterial species Enterobacter sakazakii by Farmer et al. (1980); it was later classified by Iversen et al. (2008) and Joseph et al. (2012a,b) as Cronobacter spp. and included seven species: C. sakazakii, C. malonaticus, C. universalis, C. turicensis, C. muytjensii, C. dublinensis, and C. condimenti.

Cronobacter spp. is considered as an emerging pathogen that is especially aggressive in hypersensitive individuals, such as children and the elderly (Fanning and Forsythe, 2008; Hunter and Bean, 2013). Newborns and the elderly are the population groups that are most affected by C. sakazakii, although the highest incidence and severity occurs in preterm infants (Hariri et al., 2013). Outbreaks have generally been the most common cause of infection (Jason, 2012). Clinical symptoms are mainly found in meningitis, septicemia, or necrotizing enteritis in infants (Block et al., 2002; Bowen and Braden, 2006), but diarrhea, urinary tract infection, and septicemia have also been observed. Mortality rates are associated with general infection (42–80%) and neonatal meningitis and septicemia (15–25%) (Holý and Forsythe, 2014).

The disease is associated with the consumption of rehydrated milk as a carrier of the pathogen, as well as the eventual involvement of utensils and equipment as reservoirs (Food and Agriculture Organization and World Health Organization [FAO/WHO], 2008; Kalyantanda et al., 2015). Since it is widespread, it can be isolated in powdered infant formula (PIF), rehydrated milk (R-PIF), infant cereals, various foods, water, surfaces, homes, and hospitals (Baumgartner et al., 2009). Even when the source of primary contamination is unclear (Norberg et al., 2012), some researchers suggest that the natural habitat is PIF manufacturing plants. This situation has been reported by Jacobs et al. (2011) and Li et al. (2014), who identified Cronobacter spp. in different parts of PIF plants in both China and Australia. It has also been strongly associated with by-products used in its formulation, which are also probable carriers (Jongenburger et al., 2011). The control of this pathogen in the first stages of PIF production is the most important step to reduce its incidence in the final product (Yan et al., 2012; Fei et al., 2015) because viable Cronobacter spp. strains have been found 2 years after the product was packaged (Caubilla-Barron and Forsythe, 2007).

Although updated detection and identification techniques are being used, there are still cases of disease and mortality every year (Norberg et al., 2012). It is therefore necessary to improve hygiene and the production process to reduce the impact of C. sakazakii. Biochemical tests (API 20E, RAPID, BIOLOG microarray), molecular confirmation of the Cronobacter spp. genus by polymerase chain reaction (PCR) (Lehner et al., 2004; Cetinkaya et al., 2012), and especially multilocus sequence typing (MLST) have been used to complement its identification. These techniques have allowed advances in correctly identifying it, and thus decreasing the possibility of false negatives (Baldwin et al., 2009; Joseph and Forsythe, 2011; Yan et al., 2015; Ogrodzki and Forsythe, 2017). Several primers have been generated to detect Cronobacter spp. by amplifying specific sequences of variable and conserved regions of 16S ribosomal rDNA of the bacterium (Lehner et al., 2004; Hassan et al., 2007), OmpA (Mohan-Nair and Venkitanarayanan, 2006), as well as others that are more specific, which detect C. sakazakii by rpoB (Stoop et al., 2009; Lehner et al., 2012; Li et al., 2016) and cgcA gene amplification (Carter et al., 2013).

Studies of Cronobacter spp. incidence in powdered milk have demonstrated a positivity range between 3 and 30% (Chap et al., 2009; Siqueira-Santos et al., 2013; Fei et al., 2017). Sáez et al. (2012) found 5% positivity of Cronobacter spp. in 80 PIF samples from a dairy processing plant in the Los Lagos Region in Chile. Parra-Flores et al. (2015a) found an incidence of 9.5% C. sakazakii in an exploratory study with a limited number of samples using MLST in PIF manufactured in Chile in 2014. If PIF samples manufactured in other countries are considered, incidence was 2.7% in all the analyzed PIF samples.

Given the need to ensure safety in PIFs, the FAO/WHO have held expert meetings to study cases of diseases related to its consumption, whether epidemiologically or microbiologically. Three categories of microorganisms were identified based on the soundness of evidence of a causal relationship between their presence in food and the disease: (A) microorganisms with clear causality evidence, enteric Salmonella, and Cronobacter spp. (E. sakazakii); (B) microorganisms in which causality is possible but has not yet been demonstrated, primarily from the Enterobacteriaceae family; and (C) microorganisms in which causality is less probable or has not yet been demonstrated, and have not been identified in PIF (Food and Agriculture Organization and World Health Organization [FAO/WHO], 2006; Jackson et al., 2015). The WHO therefore recommended the absence of Cronobacter spp., Salmonella, and Enterobacteriaceae in dairy products (Food and Agriculture Organization and World Health Organization [FAO/WHO], 2004, 2006).

Given that PIFs are not sterile foods, the determination of microbial indicators, such as aerobic plate count (APC) and Enterobacteriaceae (ENT), provides useful information about the hygienic conditions of their preparation or post-process contamination (Friedemann, 2009; Parra-Flores et al., 2015a; Heperkan et al., 2017).

Cronobacter spp. was not considered in the Chilean Food Sanitary Regulations (Reglamento Sanitario de los Alimentos, RSA) when the Chilean Ministry of Health decreed the food alert. The decision was taken due to the risk of disease associated with the pathogen described in the scientific literature (Jason, 2015), factors that affect PIF contamination (Parra-Flores et al., 2015b), and the variability in its cellular response (Parra-Flores et al., 2016). The PIFs associated with this alert

are not only commercialized in Chile but throughout the Americas.

Therefore, the objective of this study was to evaluate the presence of C. sakazakii and the microbiological parameters of APC and ENT in dairy products associated with a food alert in Chile in June 2017.

### MATERIALS AND METHODS

### Food Samples

Sampling was conducted from August 2016 to May 2017. Ninety samples were collected in four countries (United States, Singapore, Chile, and Holland), from three manufacturers (1, 2, and 3), seven commercial dairy brands (A, B, C, D, E, F, G) of which B, D, and F were powdered and A,C, E, and G were liquid products sold in supermarkets and pharmacies in Chile. All the analyses were performed in duplicate. The sampling criteria used as a reference were the standards of the Chilean RSA and CAC/RPC 66 of the Codex Alimentarius.

### Microbiological Quantification

The APC of the mesophilic microorganisms and ENT count were used. Quantification of both microbial groups and identification of isolated enterobacteria (including suspicious strains of Salmonella or Escherichia coli) were performed in the Accredited Food Testing and Certification Laboratory (LECYCA-UBB) and the Molecular Epidemiology and Microbiology Laboratory of the Universidad del Bío-Bío. References are NCh 2659 (2002) for AMC, NCh 2676 (2002) for ENT, NCh 2636 (2002) for E. coli, and NCh 2675 (2002) to isolate Salmonella.

### Isolation of Cronobacter spp.

The technique described by Parra-Flores et al. (2015a) was applied. For each sample, 225 mL of buffered peptone water (BPW) were added to 25 g of powdered infant formula (PIF) or dairy product (DP) and then homogenized in a stomacher at a mean velocity for 60 s. Liquid products in their original container were directly incubated at 37◦C. Then 10 mL of each sample was inoculated after incubation at 37◦C for 24 h in 90 mL Enterobacteriaceae enrichment broth (BD Difco, Sparks, MD, United States). A loop was extracted from the culture suspension and striated in Brilliance Chromogenic Agar CM 1035 (Oxoid Thermo Fisher, United Kingdom) at 37◦C for 20 h. Five strains, presumed to be colonies of Cronobacter spp. (green or blue), were striated in trypticase soy agar (BD Difco, Sparks, MD, United States) to verify their purity prior to future analyses. The isolated strains were maintained in a strain collection and stored at −80◦C.

### Identification of Cronobacter spp. and Cronobacter sakazakii

Genomic DNA of the suspicious strains was extracted and purified with the Ultra Clean R Microbial DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA, United States). The strains were confirmed as Cronobacter spp. by OmpA gene amplification (Mohan-Nair and Venkitanarayanan, 2006) and later identified as C. sakazakii by qPCR amplification of rpoB and cgcA genes (Stoop et al., 2009; Carter et al., 2013) (**Table 1**) in the Stratagene Mx3000P qPCR System equipment (Agilent Technologies).

### Sequencing of fusA Gene to Identify Species of Cronobacter spp.

The methodology described by Baldwin et al. (2009) was followed using PCR CORE Kit QIAGEN (Cat No. 201225) solutions. Amplified products were sent to MACROGEN in Korea for sequencing. Identification was performed with the free access online database https://pubmlst.org/cronobacter/ and BLASTn (NCBI).

### Bioinformatic and Statistical Analyses

The sequenced products were analyzed with the Gentle software and later aligned with the ClustalW software. A phylogenetic tree was constructed using the maximum likelihood method with the MEGA7 software. Statistical description included measures of central tendency, dispersion, and position for quantitative variables, while absolute frequencies and percentages were used for qualitative variables. The Mann–Whitney and Kruskal–Wallis tests were used for comparison purposes with the STATA 14 software at the significance level α = 0.05.

### RESULTS

Of the 90 analyzed Chilean and foreign samples, 79 had APC. When analyzing APC for each DP commercial brand, no

TABLE 1 | Polymerase chain reaction (PCR) primers used in the study.

significant statistical differences were found (p > 0.05). However, half of the liquid DP commercial brands contained 2.6 log CFU/mL, 2.3 log CFU/mL, 1.1 log CFU/mL, and 2.9 log CFU/mL for brands A, C, E, and G, respectively. The powdered DP (including PIF) commercial brands had values of 3.0 log CFU/g, 3.6 log CFU/g, and 5.7 log CFU/g for brands B, D, and F, respectively. Positivity for ENT was found in all the evaluated brands. In the liquid DP brands A, C, E, and G, the sample means were 1.8, 0.8, <0.1, and <0.1 log UFC/mL, respectively. In half of powdered DP brands B, D, and E, samples contained 2.9, 2.8, and 2.7 log CFU/g, respectively. Statistical differences were only found in the ENT counts (p = 0.012). For DP manufacturers, company 2 had the highest count with 5.9 log CFU/g. Enterobacteriaceae counts were found in 55% of the total analyzed samples. Company 1 obtained the highest counts with 3.5 log CFU/g (**Table 2**).

Regarding the country of origin (**Table 3**), Chile exhibited the highest APC count means with 5.0 log CFU/g and the United States showed the lowest count with 2.1 log CFU/g. The DP produced in Holland had the highest ENT counts followed by Singapore with 3.6 and 3.2 log CFU/g, respectively. The lowest count was obtained in the US with 0.1 log CFU/g. No significant differences existed in the APC and ENT counts for country of origin (p > 0.05). As for the type of DP, 100% of the liquid DP brands contained APC and 55% ENT counts. Only the liquid DP brands produced in the US had ENT counts with values between 0 and 4.5 log CFU/mL. The highest count in powdered DPs was obtained in Holland with 4.4 log CFU/g.

Of the total analyzed samples, 17 suspicious strains were isolated from the chromogenic agar. Nine were identified as E. cloacae, Klebsiella pneumoniae, E. hormaechei, and

TABLE 2 | Aerobic plate count (APC) and Enterobacteriaceae (ENT) count for each commercial brand and manufacturer.

∗p-value according to Kruskal–Wallis test. Brands A, C, E, and G: liquid dairy products; Brands B, D, and F: powdered milk products; ns: non-significant.

TABLE 3 | Aerobic plate count (APC) and Enterobacteriaceae (ENT) count for each country.

∗p-value according to Kruskal–Wallis test; ∗∗p-value according to Mann–Whitney test. ns, non-significant.

Enterobacter spp., whereas Salmonella spp. was not isolated in any of the samples. However, E. coli was identified in one powdered milk (PM) product manufactured in Chile.

Only eight suspicious strains from the PM from Chile and Singapore were confirmed as Cronobacter spp. by amplifying the ompA gene. These strains were subsequently confirmed as C. sakazakii through the amplification of the gene products for rpoB and cgcA by PCR in real time. One of the PM products in which C. sakazakii was isolated was intended for consumption by infants under 2 years (CH84), and another from Singapore was intended for consumption by children older than 1 year (CH65).

Furthermore, six more strains were confirmed, which were not part of the food alert because of their expiry date and were PM products manufactured in Chile (CH42, CH43, CH44, CH45, CH50, and CH85) (**Table 4**). Two more samples were also detected by real-time PCR with Cronobacter spp. from PIFs manufactured in Holland, but it was not possible to recover the pathogen from the samples.

Cronobacter sakazakii incidence in the total evaluated samples was 8.8% (**Table 5**).

All the C. sakazakii strains were genotyped by sequencing the fusA gene using MLST in the database https://pubmlst. org/cronobacter/ and BLASTn (NCBI). The information of the sequences was later used to construct a phylogenetic tree (**Figure 1**).

### DISCUSSION

The PIFs analyzed in the present study are commercialized throughout the Americas. Therefore, evaluating their microbiological quality allows determining aspects such as the hygienic conditions in which they were prepared, as well as identifying microbial hazards from probable recontamination occurring when they are supplemented with nutrients after pasteurization (Kent et al., 2015).

For APC, 72% of all analyzed powdered and liquid DP samples contained less than 3 log CFU. There were 11% of PM brands that contained between 4.0 and 4.7 log CFU/g originating only from the United States and Singapore; two products manufactured in Chile had values of 6.7 log CFU/g. Liquid DPs revealed five

TABLE 4 | Identification of Cronobacter spp. and Cronobacter sakazakii by molecular amplification.

TABLE 5 | Positivity of Cronobacter sakazakii for country of origin and product type.

NE, not evaluated.

samples that ranged between 5.6 and 5.8 log CFU/g, four of which were produced in the United States and one in Chile. These APC values are within ranges reported by other authors (Iversen et al., 2004; Kim et al., 2011; Parra-Flores et al., 2015a). However, it was a concern to find counts greater than 5 log CFU/g in PM; these values were very high compared to results reported by Chap et al. (2009), who found 2% in this range. Heperkan et al. (2017) found values between 1.7 log for PIF and 4.9 log CFU/g for PM in a study of 80 PM samples; counts were similar to those determined in the present study. There is an evident need to control the contamination sources of DM products due to the wide range of microorganisms present in the APC and the higher susceptibility of infection in children of different ages who consume DPs (Chap et al., 2009).

Positivity was found for ENT in 55% of all analyzed samples, and there was a significant statistical relationship in the counts for commercial brand (p = 0.012) and manufacturer (p = 0.010). Eight PM and two liquid DPs obtained count means of 2 log CFU and 11 samples had values from 3.4 to 4.5 log CFU. Muytjens et al. (1988) encountered ENT in 52% of 141 evaluated formulas from 35 countries. On the other hand, ENT was found in 47% of PIF manufactured in Indonesia and Malaysia (Estuningsih et al., 2006), 22.5% in Ivory Coast (Yao et al., 2012), and 100% in Chile (Parra-Flores et al., 2015a). All these ENT counts are much higher than the values permitted according to the international standard of Codex Alimentarius Commission

(2008), which requires the absence of this indicator in 10 g. In the present study, the high ENT positivity is compatible with the presence of several opportunistic microorganisms and pathogens associated with disease in infants reported in different publications (Food and Agriculture Organization and World Health Organization [FAO/WHO], 2006; Kent et al., 2015). Therefore, these findings should be analyzed in terms of risk associated with the consumption of PM by infants, the lack of control by manufacturers, and health authorities responsible for inspection in Chile. Although the association between the risk of falling ill with the consumption of ENT-contaminated PM has not yet been established with certainty, its absence in PM provides additional protection for newborns, especially the preterm, immunocompromised, and those with low (<2,500 g) and very low (<1,500 g) birth weight during the preparation, storage, and administration of infant feeding (Abdullah Sani et al., 2013).

Other microorganisms belonging to this family were also identified in the present study. E. cloacae, K. pneumoniae,

and E. hormaechei were found. This situation does not seem altogether exceptional considering that other authors have also found these microorganisms of the ENT family in PIF (Iversen et al., 2004; Giammanco et al., 2011; Kim et al., 2011; Abdullah Sani et al., 2013), and especially for the risk associated with its ability to maintain itself for at least 8 months under desiccation conditions (Caubilla-Barron and Forsythe, 2007; Juma et al., 2016). Furthermore, the Food and Agriculture Organization and World Health Organization [FAO/WHO] (2004, 2006) also recognized that other ENT can be recovered from PIF and could present a risk to infants, although no reported cases had been confirmed at that time. Jackson et al. (2015) re-evaluated a reported C. sakazakii outbreak through the consumption of contaminated reconstituted PIF in Mexico. Using DNA sequencing, they demonstrated that the causative agents were misidentified strains of E. hormaechei and Enterobacter spp. Meanwhile, E. hormaechei has been shown to have clinical significance with the report of several outbreaks of sepsis in neonatal intensive care units in Brazil and the United States (Wenger et al., 1997; Campos et al., 2007; Townsend et al., 2008).

The high APC and ENT values can indicate a non-strict adherence to hygienic practices recommended for the preparation of PIF, which has been mentioned by other authors (Mullane et al., 2007; Jongenburger et al., 2011). This can imply a permanent risk for populations that usually consume this product.

The C. sakazakii incidence was 8.8% in the total of evaluated samples, particularly in 10 and 35% of samples produced in Singapore and Chile, respectively. This high positivity should give rise to greater control by the manufacturers and health authorities because of its high lethality, related neurological sequela, and risk of falling ill by C. sakazakii (Lai, 2001; Holý and Forsythe, 2014). An infection rate of 1 in 100,000 newborns has been estimated in the United States; this rate increases to 8.7 in 100,000 in infants weighing less than 1500 g, and 1 in 10,660 preterm infants with low birth weight (Hunter and Bean, 2013). In Holland, Cronobacter spp. causes from 0.5 to 0.7% of all the cases of meningitis in infants, with a probable range of infection of 0.00062 to 0.62 cases per year. When this probability is adjusted with all the cases that have occurred in the last 30 years, the projected probability is 0.53 cases of infection per year with a rate of 1 in 100,000 infants (Reij et al., 2009). There is no doubt that these values are greatly underestimated (Kucerova et al., 2011; Jason, 2015). Patrick et al. (2014) stated that the median age in adults is 59 for disease caused by Cronobacter spp., this value has been widely referred to by the lay press and the representatives of formula manufacturers. Parra-Flores et al. (2016) evaluated cell response variability of C. sakazakii after mild heat treatments using stochastic approaches and reported that these can better describe microbial single cell response than deterministic models. They found that the mean probability of illness from the initial inoculum size of 1 cell was less than 0.2 in all cases, while the mean probability of illness was greater than 0.7 in most cases for the inoculum size of 50 cells.

A principal aspect of our study was the correct identification of Cronobacter spp. and C. sakazakii species by several methods described in the literature (**Table 1**). When comparing the methods, a very good correlation was found for these methods by using different primers with fusA gene sequencing, which today enables the most accurate speciation because it follows the whole genome phylogeny and adjusts to taxonomic changes (Forsythe et al., 2014; Jackson et al., 2014; Xu et al., 2014; Alsonosi et al., 2015; Vojkovska et al., 2016). Therefore, the information generated when using molecular techniques can improve the confidence level in the identification and confirmation of presumptive strains even when molecular tests provide the best identification and phenotyping methods (Joseph et al., 2013; Jackson and Forsythe, 2016).

The robustness of the results was a primary facet in our decision to declare the national and international food alert and the massive recall of the products involved. There was another important point health authorities needed to consider, that is, the 2007 WHO recommendation to use water at >70◦C to rehydrate PM to limit the risk of infection by Cronobacter spp. (World Health Organization [WHO], 2007). It was also recommended that rehydrated PM for children be administered within 2 h of its preparation or conservation under refrigeration at <4 ◦C. This was the main idea of the publicity campaign launched by the authorities for the Chilean population.

Unfortunately, there are situations that warn us that microbiological control cannot be relaxed in food products consumed by hypersensitive populations, such as children and the elderly. For example, the case of the recall of C. sakazakiicontaminated PM destined for children in Argentina in 2015, and the recent contamination of Lactalis milk with Salmonella spp., which affected 83 European countries. However, recent studies demonstrate that the microbiological quality of these products is still inadequate even when we know that milk and DPs for child feeding are not sterile (Parra-Flores et al., 2015a).

In summary, an inadequate microbiological quality of powdered and liquid PM consumed by children under 10 was found in the present study. The presence of ENT and C. sakazakii was also identified, which is a wake-up call to manufacturers and public health regulatory authorities in Chile and throughout the Americas. It is therefore necessary to establish greater control of hygienic conditions in PM production and microbiological vigilance to prevent unnecessary risks for the child population that massively consumes these products (Koletzko et al., 2012). Disease caused in children as a consequence of any pathogen present in the PM they consume, requires manufacturers and health authorities to ensure the highest possible level of food safety (Food and Agriculture Organization and World Health Organization [FAO/WHO], 2006; Jason, 2015; Kent et al., 2015).

### CONCLUSION

Powdered infant milk formulas (PIF) are not sterile products; according to the specifications established by the Codex Alimentarius, this type of product should be treated as a possible

food safety issue for high risk populations, such infants and neonates, due to the presence of the C. sakazakii pathogen. A total of 11% of the powdered milk brands contained APCs between 4.0 and 4.7 log CFU/g, which is considered as the rejection level by the updated Chilean Food Sanitary Regulations (RSA). Of all the samples, 55% contained Enterobacteriaceae; E. cloacae, E. hormaechei, and K. pneumoniae were identified. The overall incidence of C. sakazakii was 8.8%, which was found in samples produced either in Chile or Singapore.

Based on this information, the Chilean Ministry of Health decreed a national and international food alert and recalled all the product batches from supermarkets and pharmacies that tested positive in the study. After the first survey conducted for PIF contaminated with Cronobacter spp., it was pointed out that this microorganism was present and represented a risk that was not considered in the Chilean food safety standards. The RSA therefore included a new regulation for Cronobacter spp. in PIF in November 2017 because of social media pressure and the scientific results provided by our team.

### REFERENCES

### AUTHOR CONTRIBUTIONS

JP-F conceived the experiments. JP-F, FC-L, and JA designed the experiments. NV-R, AC, and JP-F conducted the laboratory work. JP-F, NV-R, AR, and FC-L drafted the manuscript. All authors reviewed and approved the final manuscript.

### FUNDING

This study was provided by the Research Directorate of the Universidad del Bío-Bío, Projects 161720 3/R, 091824/R, and GI 171220/EF.

### ACKNOWLEDGMENTS

We wish to thank the Chilean Ministry of Health, the Chilean Institute of Public Health, and the Research Directorate of the Universidad del Bío-Bío.

for infant nutrition. Rev. Chil. Nutr. 42, 83–89. doi: 10.4067/S0717-<PHONE_NUMBER>0100011

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Parra-Flores, Cerda-Leal, Contreras, Valenzuela-Riffo, Rodríguez and Aguirre. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Exploration of the Regulatory Mechanism of Secondary Metabolism by Comparative Transcriptomics in Aspergillus flavus

Guangshan Yao, Yuewei Yue, Yishi Fu, Zhou Fang, Zhangling Xu, Genli Ma and Shihua Wang\*

Fujian Key Laboratory of Pathogenic Fungi and Mycotoxins, Key Laboratory of Biopesticide and Chemical Biology of Ministry of Education, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China

Mycotoxins cause a huge threaten to agriculture, food safety, and human and animal life. Among them, aflatoxins (AFs) have always been considered the most potent carcinogens, and filamentous fungi from Aspergillus genus are their major producers, especially A. flavus. Although the biosynthesis path of these chemicals had been wellidentified, the regulatory mechanisms controlling expression of AF gene cluster were poorly understood. In this report, genome-wide transcriptome profiles of A. flavus from AF conducing [yeast sucrose media (YES)] and non-conducing [yeast peptone media (YEP)] conditions were compared by using deep RNA sequencing (RNA-seq), and the results revealed that AF biosynthesis pathway and biosynthesis of amino acids were significantly upregulated in YES vs. YEP. Further, a novel LaeA-like methyltransferase AFLA\_121330 (Lael1) was identified for the first time, to play a specific role in the regulation of AF biosynthesis. Contrary to LaeA, which gene deletion reduced the level, lael1 deletion resulted in a significant increase in AF production. Further, coexpression network analysis revealed that mitochondrial pyruvate transport and signal peptide processing were potentially involved in AF synthesis for the first time, as well as biological processes of ribosome, branched-chain amino acid biosynthetic process and translation were co-regulated by AfRafA and AfStuA. To sum up, our analyses could provide novel insights into the molecular mechanism for controlling the AF and other secondary metabolite synthesis, adding novel targets for plant breeding and making fungicides.

Keywords: Aspergillus flavus, aflatoxin, transcriptome, LaeA-like methyltransferase, RNA-seq

### INTRODUCTION

Aflatoxins (AFs) has always been labeled as the most potent carcinogens (Squire, 1981; Amaike and Keller, 2011). In general, AF and its major producer Aspergillus flavus have been frequently detected in oil-enriched seeds such as peanuts, maize seeds, and walnuts (Bhatnagar-Mathur et al., 2015). However, a recent fear was that aflatoxin B1 (AFB1) or aflatoxin M1(AFM1) were widely detected in our daily food on a global scale, including their presence in rice and rice products in Pakistan (Iqbal et al., 2016), sugarcane juice in Egypt (Abdallah et al., 2016), sausages in Croatia

#### Edited by:

Pierina Visciano, Università degli Studi di Teramo, Italy

#### Reviewed by:

Gang Liu, Institute of Microbiology (CAS), China Massimo Reverberi, Sapienza Università di Roma, Italy

> \*Correspondence: Shihua Wang<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

Received: 04 January 2018 Accepted: 25 June 2018 Published: 07 August 2018

#### Citation:

Yao G, Yue Y, Fu Y, Fang Z, Xu Z, Ma G and Wang S (2018) Exploration of the Regulatory Mechanism of Secondary Metabolism by Comparative Transcriptomics in Aspergillus flavus. Front. Microbiol. 9:1568. doi: 10.3389/fmicb.2018.01568

**520**

(Kabak and Dobson, 2017), milk in Europe (Kumar et al., 2017b), vegetable oil (Sun et al., 2011) and Chinese traditional medicines in China (Zhao et al., 2016), and even human breast milk in Turkey (Kiliç Altun et al., 2017). An increasing body of evidences demonstrated that exposure to or ingestion of AF severely impaired human or animal health (Kumar et al., 2017b). Long time ingestion of AF-containing food or food adducts are associated with high rates in hepatocellular carcinoma (Pitt and Miller, 2017). Even worse, several lines of evidences suggested that AF uptake and hepatitis B virus synergistically induced the development of liver cancer (Kew, 2003; Aydin et al., 2015; Chu et al., 2017). Therefore, an improved understanding of AF synthesis and metabolism, and where its regulatory mechanism is urgently required, which would greatly contribute to the development of new and effective long-term management strategies to avoid the severe effects caused by these toxic chemicals.

Until now, the AF biosynthetic pathway had been essentially clarified (Roze et al., 2013), however, the regulatory mechanism that orchestrating the AF cluster gene expression remains largely unknown. Nutrient significantly impact the synthesis of AF, and yeast sucrose media (YES) containing high concentration sucrose could induce the formation of AFs, but yeast peptone media (YEP) could not (Fountain et al., 2015). Paradoxically, an earlier report found that addition of peptone in a chemical defined media increased AFs (Reddy et al., 1979). Compared to other nitrogen source, glutamine was reported to be the best one to induce the toxin production (Wang et al., 2017). As the secondary metabolism from Aspergillus, biosynthesis of AFs was affected and governed by various environmental cues and a number of proteins at multiple levels, including chromosome, transcription, and posttranslation modifications in vivo (Amaike and Keller, 2011; Amare and Keller, 2014; Calvo and Cary, 2015). A screen of sterigmatocystin (the precursor of AFs) mutants in A. nidulans lead to the identification of an essential regulator LaeA (Bok and Keller, 2004). It is well-known that LaeA, containing a methyltransferase domain, functions as a global regulator of secondary metabolism in various filamentous fungi (Bok and Keller, 2004; Kale et al., 2008; Kosalkova et al., 2009; Chettri and Bradshaw, 2016; Kumar et al., 2017a), especially toxicogenic A. flavus (Kale et al., 2008). And, a systematic transcriptome analysis revealed that LaeA affected expression of 26 of 55 secondary metabolite biosynthetic clusters in A. flavus (Georgianna et al., 2010). Recently, LlmF, a laeA-like protein, was reported to negatively regulate the production of sterigmatocystin production in A. nidulans by modulating the nuclear import of VeA in A. nidulans (Palmer et al., 2013). As in A. nidulans, a number of novel proteins containing a methyltransferases domain showed high similarity to LaeA in A. flavus. Until now, only the role of LaeA in AF synthesis had been confirmed (Bok and Keller, 2004; Kale et al., 2008), but other LaeA-like methyltransferases remains unidentified in A. flavus.

Pathway-specific and global transcription factors (TFs) were also reported to critically involve in the regulation of AF gene cluster expression. AflR, as the AF pathway-specific TF, is absolutely required for expression of most of the genes in the AF cluster (Ehrlich et al., 1999). And, AflS (previously named as AflJ) was reported to interact with, and activate AflR to exert its regulatory role (Chang, 2003). It was now well known that fungal development was closely associated with biosynthesis of secondary metabolism (Amare and Keller, 2014; Calvo and Cary, 2015). It was recently found that several TFs co-regulated the conidiation, sclerotial development and AF formation in A. flavus, including MtfA (Zhuang et al., 2016), RtfA (Lohmar et al., 2016), and NsdC (Gilbert et al., 2016). More recently, we had identified two APSES TFs AfRafA and AfStuA to be required for fungal conidial and sclerotial development, and colonization of plants. Further, loss of AfStuA completely inhibited the biosynthesis of mycotoxins including AFs and cyclopiazonic acid, and 1AfRafA reduced AF, but enhanced the cyclopiazonic acid biosynthesis (Yao et al., 2017). Therefore, it is reasonable to speculate that AfRafA and AfStuA mediate a complex regulatory network with crucial roles in multiple cellular processes in A. flavus. Undoubtedly, comprehensively identifying their downstream genes or pathways contributes to obtain novel regulators for toxin biosynthesis or fungal pathogenicity.

In the present report, we comprehensively compared the transcriptomes of A. flavus WT strains culturing in AF inducing vs. non-inducing conditions, 1AfRafA vs. WT, and 1AfStuA vs. WT. Our results revealed that amino acid biosynthesis and metabolism were significantly activated under YES relative to YEP and subjected to regulation of both AfRafA and AfStuA. Weighted correlation network analysis of differentially expressed genes reveal that AfRafA and AfStuA coordinately control novel cellular processes with potential roles in AF biosynthesis. Further, many AF regulators were found to be regulated by AfRafA or AfStuA, or both, and activated in YES vs. YEP. In addition, our comparative transcriptome suggested that expression of many SM gene clusters differentially response to AfRafA and AfStuA. Most importantly, a novel LaeA-like protein Lael1 was demonstrated to have a specific role in regulation of the AF expression.

### MATERIALS AND METHODS

### Strains and Culture Conditions

The strains used in this study including WT, 1AfRafA, and 1AfStuA were stored in our lab constructed previously (Yao et al., 2017). The strain PTS1ku701pyrG (Chang et al., 2010) was used as the recipient strain to generate the deletion mutants for gene AFLA\_121330. All A. flavus strains were cultured onto Potato dextrose agar (PDA, BD Difco, United States) to obtain mycelia and conidia, and then stored in 30% glycerol solution at −70◦C. When compared AF synthesis, YES (2% yeast extract, 15% sucrose, and 0.1% MgSO4) and GMM (Glucose 10 g/L, NaNO3 6 g, KCl 0.52 g/L, MgSO4.7H2O 0.52 g/L, KH2PO4 1.52 g/L, and added 1 mL trace elements solution per liter) with 5 mM glutamine (Shimizu

and Keller, 2001), and YEP (2% yeast extract, 15% peptone) represents the AF conducing and non-inducing conditions, respectively.

### Determination of AF Production via TLC and HPLC

Extraction and determination of AFs were performed as previously described (Yao et al., 2017). The AFs were extracted by using chloroform. TLC analysis of AFB1 was performed with the acetone:chloroform (1:9, v/v) solvent system, and AFB1 spots were displayed under ultraviolet activation at 365 nm. HPLC analysis of AFB1 was conducted by using the Waters HPLC 1525 system (Waters, United States) equipped with a MYCOTOXTM reversed-phase C18 column (5 µm, 4.6 mm × 150 mm) and a fluorescent detector (λex = 365 nm, λem = 430 nm). Firstly, the column was equilibrated in the mobile phase (water:methanol:acetonitrile, 56:22:22) at 42◦C for 1 h. Each chloroform extract was re-dissolved in methanol, filtered through a 0.22 µm nylon filter membrane, and then separated in a 100% mobile phase at a flow rate of 1.0 mL/min. The AFB1 concentration of each sample was counted by using a calibration curves and the AFB1 standard (HPLC grade) were purchased from Sigma (Sigma, Germany).

### Gene Deletion and Complementation

Extraction of genomic DNA of A. flavus and standard PCR were performed as previously described (Yu et al., 2004). The AfRafA and AfStuA deletion strains used were constructed previously (Yao et al., 2017) and the Lael1 deletion strains was generated in the present study. Double-joint PCR was used to construct a gene deletion cassette, using the pyrG gene amplified from A. fumigatus as the selectable marker. All primers that were used to amplify the 50 - and 30 -flanks were listed in **Supplementary Table S2** with the A. flavus gDNA as template. The entire gene deletion cassette was amplified with specific primers, using the 50 and 30 -flanks for gene AFLA\_121330, and pyrG mix as template. The PCR products were transformed into the protoplasts of PTS1ku701pyrG. Protoplast preparation and PEG-mediated fungal transformation were performed following previously described methods (Chang, 2008). Diagnostic PCR and RT-PCR were used to identify the positive transformants. To construct the complementation strain, an expression cassette containing promoter, coding region, and terminator was amplified by using high-fidelity pfu DNA polymerase (TransGen, Beijing, China). Purified expression cassette together with plasmid pTRI with pyrithiamine (ptrA) as the selectable marker (Chang et al., 2010), were co-transformed into the protoplast of the deleted strain. All primers used are listed in **Table 1**.

### RNA Extraction

Aspergillus flavus strains were inoculated into YES liquid media and pre-cultured for 24 h, and then transferred into fresh YES media or YEP for stationary culture at 29◦C for 48 h. The collected mycelia were frozen in liquid nitrogen, and stored under −80◦C conditions. RNA extraction was performed using an RNA reagent Kit (TRIzol reagent, Biomarker Technologies, China)

and following the protocols of the manufacture. The quality and integrity of RNA samples were determined using Nanodrop and Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, United States), respectively, while the quantity was determined with a Qubit RNA assay kit (Life Invitrogen, United States).

### RNA-seq and Enrichment Analysis of Differentially Expressed Genes

The total RNA of three biological replicates for 1AfRafA, 1AfStuA, and WT grown in YES and YEP was sequenced. Libraries were prepared according to standard protocols from Illumina Inc. (San Diego, CA, United States) and sequenced on a HiSeq 2000 platform (Novogene, Beijing, China). Lowquality reads (Phred ≤ 20) and adaptor sequences were filtered out, and the Q20, Q30, and GC content of the clean data were calculated (**Table 2**). Sequenced clean reads were mapped against predicted transcripts of the A. flavus NRRL 3357 genome1 using Tophat v2.0.4 (Trapnell et al., 2009), and only unique matches were allowed. Transcript abundance (i.e., FPKM) were estimated using the HTSeq package. Differentially expressed genes were analyzed with the DESeq package (Anders and Huber, 2010), and both a twofold change cut-off and an adjusted p-value of ≤0.05 were established as thresholds. An enrichment analysis of differential expression was performed using the GOSeq R package (Young et al., 2010). GO terms (including cellular component, molecular function, and biological process) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were classified as significantly enriched among differentially expressed genes only when their Benjamini adjusted p-values were ≤0.05. All the RNA-seq data had been stored in GEO database with an ID of GSE107025.

### Real Time Quantitative PCR

cDNA was synthesized from above mRNA sample by using the RevertAid RT Reverse Transcription Kit (Thermo Scientific, United States) following the manufacture's instruction. Real time quantitative PCR (qPCR) was performed on a PikoReal Real-Time PCR System (Thermo Scientific, Inc.). All utilized primers

1http://fungi.ensembl.org/Aspergillus\_flavus/Info/Index

#### TABLE 2 | Summary of RNA-Seq data.

fmicb-09-01568 August 4, 2018 Time: 17:57 # 4

were listed in **Table 1**. The relative expression level of each gene was calculated using the 2−11Ct method and the expression level of actin encoding gene was used as the internal control.

### Gene Co-expression Network

Co-expression network were constructed using a WGCNA R package (Langfelder and Horvath, 2008). The weighted matrix of pair-wise connection strengths (module) was built and genes were grouped into modules by hierarchical clustering. The power β was used to calculate the correlation coefficients and β = 14 was set as the saturation level for a soft threshold. These networks were then visualized using Cytoscape software.2

### Statistical Analysis

The significance of the data was tested using the Student's t-test. A p-value of ≤0.05 was considered as significantly different.

### RESULTS

### Comparison of Transcriptome of A. flavus Between AF Inducing and Non-inducing Conditions

Previous studies reported that YES strongly stimulate the biosynthesis of AF but YEP could not (Fountain et al., 2015). Since then, YES and YEP were defined as the AF conducing and non-conducing condition, respectively. In this study, we cultured the A. flavus WT strains in YES and YEP for 7 days, and the AF production was determined. As shown in **Figure 1**, YES induce a significant production of AF (11.5 ± 0.5 mg/10 mL cultures). However, no AF could be detectable in YEP, which was consistent with the previous report (Fountain et al., 2015), and confirmed that YES and YEP were AF conducing and nonconducing conditions, respectively. To understand the molecular mechanism underlying that YES but not YEP stimulates the AF biosynthesis, the transcriptome profiles of A. flavus cultured in both YES and YEP were comprehensively explored. The 48 h RNA was extracted and analyzed via high-throughput

2http://cytoscape.org/

sequencing. High reproducible RNA-seq data were obtained from three biological replicates per culturing condition with Pearson correlation coefficients above 0.96 and 0.88 for YES and YEP, respectively (**Figure 2A**). In total, we obtained >3.3G clean bases with error rate ≤0.02 for each biological repeat of both two samples via deep sequencing. Quantifying the expected number of fragments per kilobase of transcript sequence per millions base pairs sequenced (FPKM), it was found that 2374 genes were differential expressed (DGE) with fold changes ≥2 as a threshold by comparing the transcriptomes between in YES and YEP. Among them, expression of 1336 genes were up-regulated, and 1038 genes were down-regulated in YES relative to in YEP (**Figure 2B** and **Supplementary Table S1**). Considering that seventeen percentage of A. flavus genome showed differential expression in YES vs. YEP, implying that these two media activate significantly different transcriptomes.

### Genome-Wide Identification of Gene Functions and Metabolic Pathways That Are Responsive to YES Induction, Regulation by AfRafA and AfStuA

Recently, we identified two novel APSES transcription factors (TFs), AfRafA and AfStuA, as important and essential activators for AF biosynthesis, respectively (Yao et al., 2017). To comprehensively understand their function in A. flavus, it is necessary to systematically identify both gene function and cellular processes they target. RNA-seq was performed to illustrate the gene landscapes regulated by AfRafA and AfStuA. The 48 h RNA samples of 1AfRafA and 1AfStuA under the same culture conditions as WT in YES were extracted and analyzed via sequencing. High reproducible RNA-seq data were obtained from three biological replicates per strain with Pearson correlation coefficients above 0.97 and 0.92 for 1AfRafA and 1AfStuA, respectively (**Figure 2A**). In total, we obtained >3.5G clean bases with error rate ≤0.02 and Q20 > 95% for each biological repeat of any strain via deep sequencing. Furthermore, by quantifying the FPKM, we found that 1401 genes were differential expressed when the transcriptomes were compared between 1AfRafA and WT (**Supplementary Table S2**).

FIGURE 1 | Aflatoxin production in YEP and YES. Conidia of Aspergillus flavus WT strain were grown in YEP and YES liquid for 7 days, and then 1.5 mL cultures were extracted by chloroform. TLC analysis of extracts from both YES and YEP, along with an AFB1 standard.

These include 996 upregulated and 405 downregulated genes (**Figure 2C**). At the same time, a total of only 823 differentially expressed genes (DGE) were identified in 1AfStuA vs. WT with identical threshold described above, including 356 upregulated and 467 downregulated genes (**Figure 2D** and **Supplementary Table S3**), suggesting that compared to AfStuA, AfRafA might play a more wide-range regulatory role in A. flavus. The Venn diagram showed that AfRafA and AfStuA shared 262 differential expression genes (**Figure 3A**), suggesting that these two proteins play overlapping roles in cellular regulation, which is further supported by their similar roles in AF and pathogenesis described previously (Yao et al., 2017). However, 1139 genes were subjected to AfRafA-specific regulation, while AfStuA uniquely controlled 561 genes (**Figure 3A**). It was also found that 1497 DGEs only showed increased or decreased expression in YES vs. YEP, unaffected by AfRaf or AfStuA. Functional enrichment of KEGG pathway of the DGEs between the deletion and WT strains, as well as AF-conducing vs. non-conducing conditions were performed, and the results were displayed in **Figures 3B–D**. Enriched analyses of the upregulated DGEs in YES uncovered that AF biosynthesis (afv00254) was significantly enriched, which accorded with our expectation (**Figure 3B**). Furthermore, DNA replication, purine metabolism, ribosome, and biosynthesis of amino acids were also significantly activated in YES (**Figure 3B**), which implying that they might have roles in AF synthesis. In addition, the downregulated DGEs were significantly enriched in valine, leucine and isoleucine degradation, penicillin and cephalosporin biosynthesis, and alpha-linolenic acid metabolism. As expected, the most downregulated genes in both 1AfRafA vs. WT and 1AfStuA vs. WT were also responsible for the pathway of AF biosynthesis (**Figures 3C,D**), further confirming that these two TFs function as key regulators for activating this toxin biosynthesis. These results are in well agreement with our previous AF production assays (Yao et al., 2017). Likewise, a number of metabolic processes, including valine, leucine and isoleucine biosynthesis, 2-oxocarboxylic acid metabolism, and pantothenate and CoA biosynthesis, which might relate to AF and other SMs biosynthesis were downregulated in 1AfRafA vs. WT (**Figure 3C**). Correspondingly, the degradation of valine, leucine, and isoleucine was significantly upregulated in 1AfRafA vs. WT. In addition, non-homologous end-joining and homologous recombination were markedly activated in 1AfRafA, suggesting that AfRafA possibly involve in the DNA repair process. More interestingly, only AF biosynthesis was enriched in downregulated DGEs in 1AfStuA vs. WT (**Figure 3D**), confirming that AfStuA functioned as essential AF-specific regulator. On the contrary, biosynthesis of other secondary metabolites showed upregulation in 1AfStuA vs. WT, indicating a block of AF synthesis in 1AfStuA would promote the redirection of metabolic flux to synthesize other SMs.

### Co-expression Network Analysis

To explore the relationship between AfRafA and AfStuA regulons, a network analysis was determined by WGCNA (Langfelder and Horvath, 2008). According to the level of AFB1 production, we set phenotype of WT, 1AfRafA, and 1AfStuA as 1, 0.5, and 0, respectively. A gene set with differential expression were clustered into several modules labeling as distinct colors. Two modules (MEblack and MEgreenyellow) showed the highest correction with phenotype (R 2 = 0.86 and R 2 = 0.89). Three hundred and twelve genes of MEblack module were interacted to form the network **Figure 4B**. GO enrichment analysis of these genes showed that ribosome (GO:0005840), branched-chain amino acid biosynthetic process (GO:0009082) and translation (GO:0006412) were identified. Network from genes from MEgreenyellow module was shown in **Figure 4C** and no GO term was significantly enriched. No AF could be detected in YEP-cultured WT and YES-cultured 1AfStuA,it is interesting to uncover general mechanism for aflatoxigenic phenotype. Analysis of all RNA-seq data of WT\_YES, WT\_YEP, and 1AfStuA were cluster into 21 modules. One module (colored as MEmagenta) had a significant correction with phenotype (**Figure 4D**). Two hundred and eighty-seven genes from this module generated a gene co-expression network (**Figure 4E**). Among these genes, branched-chain amino acid biosynthetic process (GO:0009082), mitochondrial pyruvate

transport (GO:0006850), signal peptide processing (GO:0006465) were enriched. It was worth to note that mitochondrial pyruvate transport and signal peptide processing potentially implicated in AF biosynthesis was proposed for the first time.

### Most Up-regulated Genes in YES vs. YEP Involve in Carbon and Nitrogen Metabolism

Analyses of the top twenty in upregulated DGEs in YES vs. YEP showed that most of them have roles in the nitrogen and carbon metabolism. Two ammonium transporters genes (AFLA\_108260 and AFLA\_130040) were increased by above fivefold and sixfold under AF conducing condition compared with growth in non-conducing one. Likewise, the glutamate synthase gene (AFLA\_022340), glutamate/phenylalanine/leucine/valine dehydrogenase gene (AFLA\_113320), the amino acid transporter gene (AFLA\_073030) were also up-regulated above fivefold in the AF conducing condition. Three most upregulated genes (7- ∼8-fold) were involved in carbon metabolism including alcohol dehydrogenase (AFLA\_097820), carbohydrate kinase (AFLA\_097830), glyceraldehyde 3-phosphate dehydrogenase

(AFLA\_042390). In addition, lipopolysaccharide-modifying protein (AFLA\_002000), centromere protein V (AFLA\_096180), isoprenoid synthase (AFLA\_042370), O-methyltransferase (AFLA\_016120), a TF with winged helix-turn-helix DNA-binding domain (AFLA\_016130), antibiotic biosynthesis monooxygenase (AFLA\_121090), phosphoesterase (AFLA\_050610), and five genes with unknown function were identified, their roles in AF biosynthesis required further characterization. To summarize briefly, gene expression profiles of A. flavus strain cultures from YES and YEP were compared, and confirmed that AF biosynthesis, and genes related with carbon and nitrogen metabolism were significantly up-regulated in YES vs. YEP.

### Monooxygenase Subject to Specific Regulation of AfStuA

Intriguingly, the monooxygenase activity (GO:0004497) was significantly enriched when comparing 1AfStuA and WT transcriptomes. In fact, 33 of 116 genes that encode cytochrome P450 monooxygenase showed differential expression (**Supplementary Table S4**). Among these, 25 genes were downregulated, and four genes were up-regulated in 1AfStuA. Remarkably, nine monooxygenase genes (aflX, aflW, aflV, aflQ, aflI, aflL, aflG, aflN, and aflCa) from the AF cluster were concurrently and strongly downregulated in 1AfStuA. These results suggest a possibility that a variety of monooxygenases targeted by AfStuA, might contribute to the defective phenotype of 1AfStuA. Consistent with this result is a recent study, which reported cytochrome P450 monooxygenases to be widely involved in various cellular processes, including fungal development, secondary metabolism, and virulence in the plant pathogen Fusarium graminearum (Shin et al., 2017). In summary, these data demonstrated that AfStuA exerted extensive regulatory roles in cellular primary and secondary metabolism, possibly by modulating the expression of various monooxygenases.

### Co-regulated Genes by AfRafA and AfStuA Are Mainly Enzymes Functioning at Early and Middle Stages in AF Biosynthesis

To fully understand the role of AfRafA and AfStuA in the activation of the AF gene cluster, the expression of 29 genes that were responsible for the generation of AF were selected out and compared as illustrated in **Figure 4**. A previous study reported that the enzymatic reactions, which are responsible for AF synthesis could be divided into three stages: early,

middle, and late stage (**Figure 5**). AflD, AflM, and AflP in A. flavus, as well as NorA, Ver-1, and OmtA in A. nidulans were representatives in the three stages, respectively (Chanda et al., 2009). Interestingly, genes encoding early enzymes showed a more severe downregulation of their transcription in both deletion strains relative to those decoding enzymes that functioning at middle and late stages, which was reflected by the almost undetectable mRNA of most of these early genes in 1AfStuA. As expected, almost all AF genes (not including AflR) were significantly induced in YES in comparison to in YEP (**Figure 5**). And, most of the AF cluster genes in this group conformed an expression pattern that was highest in WT, lower in 1AfRafA, and even lower or completely block in 1AfStuA, indicating that AfStuA played a more significant regulatory role in AF biosynthesis relative to AfRafA. However, the three late enzymes encoding genes aflW, aflX, and aflY showed a WT-level expression in 1AfRafA and 1AfStuA. In summary, AfStuA and AfRafA played key roles in activating the AF gene cluster at the initial phase, also demonstrating that expression of the earlystage enzyme genes for AF synthesis were co-regulated by these two TFs.

### Expression of Multiple AF Regulators Are Responsive to YES, or AfRafA and AfStuA Regulation

Interestingly, 12 known AF regulators were identified by our comparative transcriptome analysis, because their expression pattern showed differential response to culturing condition, and AfRafA and AfStuA regulation. Two transcription regulators MeaB and RtfA, was previously identified as negative regulators (Amaike et al., 2013; Lohmar et al., 2016), and their expression levels were significantly downregulated in YES when compared with in YEP, but seems not to be regulated by AfRafA or AfStuA in this study (**Figures 6A,B**). Unexpectedly, the bZIP TF AtfB, positively regulating AF biosynthesis (Wee et al., 2017), its transcription level was downregulated by twofold in YES vs. in YEP, whereas upregulated by threefold in 1AfStuA mutant in compassion to WT (**Figure 6C**), implying that AfStuA regulates AF in an AtfB-independent way. G protein-coupled receptor gene gprP was up-regulated in all strains used in YES compared with WT cultured in YEP (**Figure 6D**), and its encoding protein negatively control the AF biosynthesis (Affeldt et al., 2014). On the contrary, expression of two phosphodiesterase genes pdeH and pedL were downregulated significantly in YES in each mutant strain relative to YEP (**Figures 6E,F**), which was consistent with their negative roles in toxin formation (Yang et al., 2017). More recently, the novel protein LaeB was demonstrated to be crucially required for both sterigmatocystin and AFs biosynthesis in A. nidulans and A. flavus, respectively (Pfannenstiel et al., 2017). Unexpectedly, it was showed that expression of laeB was markedly upregulated in YES, and upregulated by AfStuA without statistical significance, but not by AfRafA (**Figure 6G**). Oxylipin-generating dioxygenases include PpoC, were reported to involve in the AF synthesis (Brown et al., 2009). In accordance with the expression pattern of