Disinfection efficiency of chlorine dioxide and peracetic acid against MNV-1 and HAV in simulated soil-rich wash water.

Wash water from fresh vegetables and root vegetables is an important vehicle for foodborne virus transmission. However, there is lack of assessing rapid viral inactivation strategies in wash water characterized by a high soil content at the post-harvest stage. Considering the significance of food safety during the washing stage for fresh and root vegetable produce prior to marketing, we assessed the inactivation efficacy by using chlorine dioxide (ClO2) and peracetic acid (PAA) against a surrogate of human norovirus (murine norovirus 1, MNV-1) and hepatitis A virus (HAV), in wash water containing black soil and clay loam. The results indicated that MNV-1 and HAV were reduced to the process limit of detection (PLOD), with reductions ranging from 4.89 to 6.35 log10 PFU, and 4.63 to 4.96 log10 PFU when treated with ClO2 at 2.5 ppm for 10 mins. Comparatively, when treated with 500 ppm of PAA for 10 mins, MNV-1 and HAV were maximum reduced to 1.75 ± 0.23 log10 PFU (4.50 log10 PFU reduction) and 2.13 ± 0.12 log10 PFU (2.72 log10 PFU reduction). This demonstrated the efficacy of ClO2 in eliminating foodborne viruses in soil-rich wash water. When we validated the recovery of the virus from two types of wash water, the pH (9.24 ± 0.33 and 5.95 ± 0.05) had no impact on the recovery of MNV-1, while the recovery of HAV was less than 1 %. By adjusting the pH to a neutral level, recovery of HAV and its RNA levels was increased to 15.94 and 3.89 %. Thus, this study emphasized the critical role of pH in the recovery of HAV from the complex soil-rich aqueous environment, and the efficacy of ClO2 serving as a pivotal reference for the development of control strategies against foodborne viruses in the supply chain of fresh and root vegetables.

Characterization and genomic insight of surfactin-producing Bacillus velezensis and its biocontrol potential against pathogenic contamination in lettuce hydroponics.

Due to food borne pathogen, maintaining the viability of fresh fruits and vegetable is a great concern. Several strategies including microbial and plant-based formulations to reduce their infection and maintain quality of the fresh food are in practice. Currently, Bacillus has gained significant traction as a biocontrol agent for regulating diseases affecting a variety of agricultural and horticultural crops. Food-grade citric acid and plant growth-promoting rhizobacteria (PGPR) were used as antimicrobial agent, MIC results showed that PGPR (14.87 mm) and CA (20.25 mm) exhibited notable antimicrobial activity against E. coli. Lettuce treated with PGPR showed reduction in E. coli contamination, E. coli was detected at 3.30, 3.68 in control, and 2.7 log CFU/g in random root injury lettuce inoculated with PGPR KACC 21110 respectively. Random root injury showed a trend toward increasing E. coli internalization. The strains exhibited resistance to multiple antibiotics, including Imipenem, tetracycline, ampicillin, cefotaxime, cefoxitin, and ceftriaxone. Comprehensive data analysis revealed the presence of ten putative bacteriocin or bacteriocin-like gene clusters. The structure of lipopeptide homologs was characterized by using QTOF-MS/MS. The mass ion peaks attributed to surfactin homologs, surfactin A ion at m/z 1008.66, surfactin B, C at m/z 1022.67 and 1036.69. In addition to surfactin, a polyketide oxydifficidin and lipopeptide NO were extracted and detected from the extract of B. velezensis. Both isolates are key biocontrol agents and have significant potential in combating foodborne pathogens and can be utilized to explore novel antibacterial products for preventing pathogens in fresh produce.

Identification and Characterization of Antibiotic-Resistant, Gram-Negative Bacteria Isolated from Korean Fresh Produce and Agricultural Environment.

The consumption of fresh produce and fruits has increased over the last few years as a result of increasing consumer awareness of healthy lifestyles. Several studies have shown that fresh produces and fruits could be potential sources of human pathogens and antibiotic-resistant bacteria. In this study, 248 strains were isolated from lettuce and surrounding soil samples, and 202 single isolates selected by the random amplified polymorphic DNA (RAPD) fingerprinting method were further characterized. From 202 strains, 184 (91.2%) could be identified based on 16S rRNA gene sequencing, while 18 isolates (8.9%) could not be unequivocally identified. A total of 133 (69.3%) and 105 (54.7%) strains showed a resistance phenotype to ampicillin and cefoxitin, respectively, while resistance to gentamicin, tobramycin, ciprofloxacin, and tetracycline occurred only at low incidences. A closer investigation of selected strains by whole genome sequencing showed that seven of the fifteen sequenced strains did not possess any genes related to acquired antibiotic resistance. In addition, only one strain possessed potentially transferable antibiotic resistance genes together with plasmid-related sequences. Therefore, this study indicates that there is a low possibility of transferring antibiotic resistance by potential pathogenic enterobacteria via fresh produce in Korea. However, with regards to public health and consumer safety, fresh produce should nevertheless be continuously monitored to detect the occurrence of foodborne pathogens and to hinder the transfer of antibiotic resistance genes potentially present in these bacteria.

Simulation of contamination and elimination of Escherichia coli, Listeria monocytogenes, and Murine norovirus 1 (MNV-1) from the washing process when handling of potatoes.

Root vegetables, which are in close contact with soil, are particularly vulnerable to soil contamination or decay as they can be contaminated from multiple sources, including primary production and processing. This study investigated effective washing conditions to reduce the microbial contamination of potatoes by using soaking and shaking in the washing process. The reduction of Escherichia coli, Listeria monocytogenes, and Murine norovirus 1 (MNV-1) in four washing processes (soaking only, shaking only, combined soaking-shaking I, and combined soaking-shaking I-shaking II) were compared. The numbers of E. coli and L. monocytogenes decreased by 0.55 and 0.49 log CFU/g after shaking only, 1.96 and 1.80 log CFU/g after soaking, 2.07 and 1.67 log CFU/g after soaking-shaking I, and 2.42 and 1.90 log CFU/g after soaking-shaking I-shaking II, respectively. The combined process reduced the microbial contamination more efficiently than shaking only. The reduction of E. coli in the washing process was higher than that of L. monocytogenes by approximately 0.5 logs. MNV-1 showed a reduction in the soaking and shaking steps by 1.34 and 1.98 log GC/100 g, with no significant reduction observed after the combination process. A combined process of soaking-shaking I-shaking II was effective to eliminate E. coli, L. monocytogenes, and MNV-1 from potatoes during the handling and washing process.

Combination of paper membrane-based filtration and ultrafiltration to enhance the detection of MNV, HAV, and HCoV from soil-rich post-washing water.

Risk-assessing and controlling virus transmission from soil-rich post-washing water (PWW) are crucial during harvesting raw vegetables. However, viruses are normally difficult to concentrate because of their low concentrations and complex backgrounds. Here, ultrafiltration (UF), virus adsorption-elution (VIRADEL), and optimized paper filtration-coupled ultrafiltration (PFC-UF) methods were employed to evaluate the recovery of non-enveloped murine norovirus (MNV-1), hepatitis A virus (HAV), and enveloped human coronavirus 229E (HCoV-229E) from soil-rich PWW. Among the three methods, PFC-UF outperformed the other methods in the recovery of viruses from PWW with soil content. Under the highest soil condition with virus seeded at a titer of 102 plaque-forming unit (PFU) or TCID50, the PFC-UF method exhibited an exceedingly consistent recovery rate of 78.8 ± 13.3 (MNV-1) and 44.4 ± 25.2% (HAV). However, the recovery of enveloped HCoV-229E was inferior to non-enveloped viruses. Overall, PFC-UF provided a reliable method for recovering viruses in soil-rich PWW.

Prevalence, distribution, enterotoxin profiles, antimicrobial resistance, and genetic diversity of Bacillus cereus group isolates from lettuce farms in Korea.

Lettuce wraps are popular in Korean cuisine for their high nutritional value and versatility as healthy additions to multiple dishes. Microbial contamination of lettuce is a major concern, as lettuce is consumed fresh without cooking. Among foodborne pathogens, the spore-forming, facultative anaerobic bacterium, Bacillus cereus is one of the frequently detected pathogen in lettuce in Korea. In this study, we investigated the prevalence and distribution of Bacillus cereus strains in lettuce production farms and further evaluated the enterotoxin gene profiles, antibiotic susceptibility, multidrug resistance pattern, and genetic differences among the B. cereus group isolates. Of the 140 samples isolated from 10 lettuce production farms, 30 samples (21.42%) were positive for B. cereus in which 19 (31.6%) and 10 (23.25%) were from soil and lettuce, respectively. The enterotoxin patterns A (hblCDA, nheABC, entFM, and cytK genes) and B (hblCDA, nheABC, and entFM genes) accounted for 50% and 20% of all the isolates, whereas the emetic gene cesB was not detected in any of the B. cereus group isolates. Antibiotic susceptibility testing of the B. cereus group isolates revealed that all the strains were predominantly resistant to ß-lactam antibiotics except imipenem and generally susceptible to most of the non ß-lactam antibiotics, including gentamycin, streptomycin, chloramphenicol, and tetracycline. ERIC-PCR and MLST analysis revealed high genetic diversity among the 30 B. cereus group isolates, which belonged to 26 different sequence types (STs) and seven new STs. Moreover, isolates with identical STs exhibited similar patterns of antibiotic resistance and enterotoxin profiles. Results of this study indicate a high prevalence of B. cereus group isolates in lettuce production farms in the Republic of Korea.

Assessment of human norovirus inhibition in cabbage kimchi by electron beam irradiation using RT-qPCR combined with immunomagnetic separation.

Cabbage Kimchi, a traditional Korean fermented food, has occasionally been related to acute gastroenteritis caused by human norovirus (HuNoV). The present study examined the inhibitory effects of electron beam (e-beam) irradiation (1, 3, 5, 7, and 10 kGy) on HuNoV GII.4 in suspension or cabbage Kimchi using reverse transcription quantitative polymerase chain reaction combined with immunomagnetic separation (IMS/RT-qPCR). In addition, physicochemical and sensorial analyses were conducted to assess any change in the quality of cabbage Kimchi following e-beam irradiation. Following e-beam irradiation at 1 to 10 kGy, HuNoV significantly decreased to 0.28 to 2.08 log10 copy number/mL in suspension (P < 0.05). HuNoV levels in cabbage Kimchi were also significantly reduced to 0.26 to 1.57 log10 copy number/mL following irradiation with 1 to 10 kGy (P < 0.05) compared to positive control (6.0 log10 copy number/mL). The D-values for 1 log10 reduction (90% inhibition) of HuNoV in suspension and cabbage Kimchi were 4.94 and 6.96 kGy of e-beam, respectively. The pH and acidity in the irradiated cabbage Kimchi were 4.41 to 4.58 and 0.61% to 0.71%, respectively, indicating that e-beam did not affect the optimal pH or acidity. Although a slight increase of greenness was observed in the leaf portion of cabbage Kimchi irradiated with 7 to 10 kGy of e-beam, this color change was minimal and went undetected by panelists in the sensorial evaluation. The five properties of sensorial quality assessed were no different in the irradiated Kimchi sample compared with the control sample (nonirradiated cabbage Kimchi). Therefore, this study suggests that ≥6.96 kGy of e-beam could be applied in the cabbage Kimchi industry to obtain >90% of HuNoV without affecting the quality. PRACTICAL APPLICATION: As the most representative food in Korea, Kimchi needs the sanitation technology that can inhibit viral infection. Our findings suggest that e-beam irradiation can be used to reduce HuNoV effectively in Kimchi without changes in sensorial quality.

Application of high hydrostatic pressure for the inactivation of norovirus and quality stability in fresh sea squirt (Halocynthia roretzi).

Sea squirt (Halocynthia roretzi) is considered a potential cause of human norovirus in Korea. This study investigated the effect of high hydrostatic pressure at 100-500 MPa for 5 min at room temperature (23 ± 2 ℃) on the inactivation of murine norovirus-1 (initial inoculum of 6-7 log10 plaque forming units/ml) as a human norovirus surrogate in fresh sea squirt. The effects of high hydrostatic pressure on the Hunter colors and pH were also examined as the main indices of quality. No reductions in murine norovirus-1 titers were observed in sea squirt treated at 100-400 MPa. However, murine norovirus-1 in sea squirt was completely inactivated by 500 MPa of high hydrostatic pressure. Furthermore, the Hunter colors ("L," "a," and "b") and pH values (6.10-6.19) were not significantly (P > 0.05) different between non-high hydrostatic pressure-treated sea squirts and all high hydrostatic pressure-treated sea squirts. Therefore, 500 MPa of high hydrostatic pressure at room temperature may be an optimal treatment for Tunicata meat without altering the food quality (color and pH).