Eun-Jung Roh

Microbiota on Spoiled Vegetables and Their Characterization

Spoilage causes vegetables to deteriorate and develop unpleasant characteristics. Approximately 30 % of fresh vegetables are lost to spoilage, mainly due to colonization by bacteria. In the present study, a total of 44 bacterial isolates were obtained from a number of spoiled vegetables. The isolates were identified and classified into 20 different species of 14 genera based on fatty acid composition, biochemical tests, and 16S rDNA sequence analyses. Pseudomonas spp. were the species most frequently isolated from the spoiled vegetables. To evaluate the spoilage ability of each species, a variety of fresh vegetables were treated with each isolate and their degree of maceration was observed. In addition, the production of plant cell wall-degrading enzymes (PCWDEs), such as cellulase, xylanase, pectate lyase, and polygalacturonase, was compared among isolates to investigate their potential associations with spoilage. Strains that produce more PCWDEs cause spoilage on more diverse plants, and pectinase may be the most important enzyme among PCWDEs for vegetable spoilage. Most gram-negative spoilage bacteria produced acylated homoserine lactone, a quorum-sensing signal molecule, suggesting that it may be possible to use this compound effectively to prevent or slow down the spoilage of vegetables contaminated with diverse bacteria.

Characterization of a New Bacteriocin, Carocin D, from Pectobacterium carotovorum subsp. carotovorum Pcc21

ABSTRACT Two different bacteriocins, carotovoricin and carocin S1, had been found in Pectobacterium carotovorum subsp. carotovorum, which causes soft-rot disease in diverse plants. Previously, we reported that the particular strain Pcc21, producing only one high-molecular-weight bacteriocin, carried a new antibacterial activity against the indicator strain Pcc3. Here, we report that this new antibacterial activity is due to a new bacteriocin produced by strain Pcc21 and named carocin D. Carocin D is encoded by the caroDK gene located in the genomic DNA together with the caroDI gene, which seems to encode an immunity protein. N-terminal amino acid sequences of purified carocin D were determined by Edman degradation. In comparison with the primary translation product of caroDK, it was found that 8 amino acids are missing at the N terminus. This finding proved that carocin D is synthesized as a precursor peptide and that 8 amino acids are removed from its N terminus during maturation. Carocin D has two putative translocation domains; the N-terminal and C-terminal domains are homologous to those of Escherichia coli colicin E3 and Pseudomonas aeruginosa S-type pyocin, respectively. When caroDK and caroDI genes were transformed into carocin D-sensitive bacteria such as Pcc3, the bacteria became resistant to this bacteriocin. Carocin D has one putative DNase domain at the extreme C terminus and showed DNase activity in vitro. This bacteriocin had slight tolerance to heat but not to proteases. The caroDK gene was present in only 5 of 54 strains of P. carotovorum subsp. carotovorum. These results indicate that carocin D is a third bacteriocin found in P. carotovorum subsp. carotovorum, and this bacteriocin can be readily expressed in carocin D-sensitive nonpathogenic bacteria, which may have high potential as a biological control agent in the field.

Survival of Salmonella enterica and Listeria monocytogenes in Chicken and Pig Manure Compost

Livestock manure is a valuable source of nutrients and organic matter for plant. Thus, livestock manure compost is commonly used fertilizer in organic vegetable and fruit production in many countries. However, contaminated or inadequate manure compost can give negative effect to soil microorganisms. This study was conducted to investigate the survival difference of Salmonella enterica and Listeria monocytogenes in chicken and pig manure compost under the selected environmental conditions. Commercially available manure compost (pig, chicken) was inoculated with S. enterica and L. monocytogenes. Manure compost was incubated at 25℃ and consistent moisture content. Samples had been collected during 200 days depending on the given conditions. S. enterica survived for 130 days in pig manure compost and over 200 days in chicken manure compost, respectively. L. monocytogenes persisted for 120 days in pig manure compost and over 200 days in chicken manure compost, respectively. It is noted that the number of S. enterica and L. monocytogenes gradually decreased over time. The results indicate that S. enterica survived longer than L. monocytogenes in manure compost at 25℃. S. enterica and L. monocytogenes survived longer in chicken manure compost than in pig manure compost. Increased knowledge of pathogen behavior in agricultural environments is a valuable part of future work on improving risk evaluations and, in a longer perspective, in providing data for guidelines regarding safe handling of pathogen-contaminated manure compost and soil.

Prevalence of Pathogenic Bacteria in Livestock Manure Compost and Organic Fertilizer

In recent years, there has been an increasing public concern about fecal contamination of water, air and agricultural produce by pathogens residing in organic fertilizers such as manure, compost and agricultural by-products. Efforts are now being made to control or eliminate the pathogen populations at on-farm level. Development of efficient on-farm strategies to mitigate the potential risk posed by the pathogens requires data about how the pathogens prevail in livestock manure composts and organic fertilizers. Microbiological analysis of livestock manure composts and organic fertilizers obtained from 32 and 28 companies, respectively, were conducted to determine the total aerobic bacteria count, coliforms, Escherichia coli count and the prevalence of Staphylococcus aureus, Bacillus cereus, Salmonella spp., Escherichia coli O157:H7, Listeria monocytogenes, and Cronobacter sakazakii. The total aerobic bacteria counts in the livestock manure composts and organic fertilizers were in the range of 7 to and 4 to , respectively. In the livestock manure composts, coliforms and E. coli were detected in samples obtained from 4 and 2 companies, respectively, in the range of 2 to and . In the organic fertilizers, coliforms and E. coli were detected in samples obtained from 4 and 1 companies, respectively, in the range of 1 to and . In 3 out 32 compost samples, B. cereus was detected, while other pathogens were not detected. In 28 organic fertilizers, no pathogens were detected. The complete composting process can result in the elimination of pathogens in livestock manure compost and organic fertilizer. The results of this study could help to formulate microbiological guidelines for the use of compost in environmental-friendly agriculture. This research provides information regarding microbiological quality of livestock manure compost and organic fertilizer.

Genetic Diversity and Antibiotic Resistance Patterns of Staphylococcus Aureus Isolated from Leaf Vegetables in Korea

Staphylococcus aureus is an important foodborne pathogen on global basis. The current study investigated the genetic patterns in S. aureus isolates from leaf vegetables (n = 53). Additional isolates from livestock (n = 31) and humans (n = 27) were compared with the leaf vegetable isolates. Genes associated with toxins, antibiotic resistance, and pulsed-field gel electrophoresis (PFGE) patterns were analyzed. At least 1 enterotoxin-encoding gene (sea, seb, sec, sed, and see) was detected in 11 of 53 (20.75%) leaf vegetable isolates. When the agr (accessory gene regulator) grouping was analyzed, agr II was the major group, whereas agr IV was not present in leaf vegetable isolates. All S. aureus isolates from leaf vegetables were resistant to more than one of the antibiotics tested. Nineteen of 53 (35.85%) isolates from leaf vegetables exhibited multidrug-resistance, and 11 of these were MRSA (methicillin-resistant S. aureus). A dendrogram displaying the composite types of S. aureus isolates from 3 origins was generated based on the combination of the toxin genes, agr genes, antibiotic resistance, and PFGE patterns. The isolates could be clustered into 8 major composite types. The genetic patterns of S. aureus isolates from leaf vegetables and humans were similar, whereas those from livestock had unique patterns. This suggests some S. aureus isolates from leaf vegetables to be of human origin.

Monitoring of Pathogenic Bacteria in Organic Vegetables from Korean Market

This study was undertaken to assess the microbiological quality and prevalence of pathogens in organic vegetables produced in Korea. A total of 189 organically grown vegetable samples (perilla leaf 50, lettuce 50, tomato 39, cucumber 50) were analyzed for the presence of aerobic plate count, Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus, Listeria monocytogenes, and Yersinia enterocolitica. The total aerobic plate counts were in the range of 4.2 to for perilla leaf, 5.0 to for lettuce, 4.0 to for tomato, and 6.6 to for cucumber. The highest counts were found in cucumber. E. coli O157:H7, Salmonella spp., S. aureus, L. monocytogenes, and Y. enterocolitica were not detected from any organically grown vegetable samples. This research suggests that continuous monitoring in organic vegetables is required to improve fresh produce safety.

Changes of Bacterial Diversity Depend on the Spoilage of Fresh Vegetables

Almost 10~30% of vegetables were discarded by the spoilage from farms to tables. After harvest, vegetables are often spoiled by a wide variety of microorganisms including many bacterial and fungal species. This investigation was conducted to extent the knowledge of relationship the spoilage of vegetables and the diversity of microbes. The total aerobic bacterial numbers in fresh lettuce, perilla leaf, and chicory were , , of fresh weight, respectively. The most common bacterial species were Pseudomonas spp., Alysiella spp., and Burkholderia spp., and other 18 more genera were involved in. After one week of incubation of those vegetables at , the microbial diversity had been changed. The total aerobic bacterial numbers increased to , , and of fresh weight for lettuce, perilla leaf, and chicory that is about times increased bacterial numbers than that before spoilage. However, the diversity of microbes isolated had been simplified and fewer bacterial species had been isolated. The most bacterial population (~48%) was taken up by Pseudomonas spp., and followed by Arthrobacter spp. and Bacillus spp. The spoilage activity of individual bacterial isolates had been tested using axenic lettuce plants. Among tested isolates, Pseudomonas fluorescence and Pantoea agglomerans caused severe spoilage on lettuce.

Genus-specific distribution and pathovar-specific variation of the glycinecin R gene homologs in Xanthomonas genomes

Xanthomonas axonopodis pv. glycines produces bacteriocins called glycinecin, and two glycinecin genes, glyA and glyR, were reported previously. In this paper, we describe genomic distribution and variation of the glyR gene revealed by extensive Southern hybridization analysis. In contrast to the glyA gene present only in X. axonopodis pv. glycines, the glyR gene was found to be distributed widely in all the pathovars of Xanthomas genus. It was also found that the glyR gene is a multigene family while the glyA is a single copy gene. Moreover, the copy number and the variation of the glyR multigene are unique to each pathovar of Xanthomonas. The uniqueness can be easily detected by the patterns resulted from Southern hybridization using the genomic digests. Thus, we suggest the glyR gene can serve as a useful genus-specific and pathovar-specific DNA marker for Xanthomonas. One of the glyR homologs was further isolated from X. axonopodis pv. glycines, and analyzed to be functional with strong inhibitory activity against several members of Xanthomonas.

Isolation of Bacteriophages Which Can Infect Pectobacteirum carotovorum subsp. carotovorum

Bacteriophages of Pectobacterium carotovorum subsp. carotovorum which causes soft rot on diverse vegetables had been isolated from 6 major Chinese cabbage cultivation areas in Korea. In order to isolate bacteriophages, total 15 different strains of P. carotovorum subsp. carotovorum isolated from nation-wide of Korea had been used as a host. When we tested 30 different soil samples individually from Pyeongchang and Taebaek with 15 different strains as a host, Taebek soil samples showed bacteriophage plaques with almost all different indicator strains but Pyeongchang soil samples showed plaques only with P. carotovorum subsp. carotovorum Pcc2 and Pcc3 strains. Especially, P. carotovorum subsp. carotovorum Pcc3 strain was able to produce plaques with almost all soil samples. Thus, this strain can be used as an indicator strain for P. carotovorum subsp. carotovorum bacteriophage screening. Electron microscope observation revealed P. carotovorum subsp. carotovorum bacteriophages isolated in Korea were belonged to three different families, Myoviridae, Siphoviridae and Podoviridae in order Caudovirales.

Suppression Effect on Soft-rot by Bacteriocin-producing Avirulent Pectobacterium carotovorum subsp. carotovorum Pcc21-M15

Pectobacterium carotovorum subsp. carotovorum causes soft rot disease in diverse plants. Carocin D is bacteriocin that is produced by Pectobacterium carotovorum subsp. carotovorum Pcc21 strain. Nonpathogenic mutant P. carotovorum subsp. carotovorum Pcc21-M15 strain was obtained by mutagenesis with Tn5 insertion and screened pathogenesity. P. carotovorum subsp. carotovorum Pcc21-M15 and E. coli (pRG3431), carocin D gene-transformed E. coli, produce carocin D against P. carotovorum subsp. carotovorum Pcc3. Pathogenic P. carotovorum subsp. carotovorum Pcc3 and mixture with Pcc21-M15 or E. coli (pRG3431) were treated with lettuces. Pcc21-M15 and E. coli (pRG3431) effectively suppressed the development of soft-rot disease. While symptoms in 90% of Pcc3-treated lettuces were observed after 3 days, only 25% of Pcc3 and Pcc21-M15-treated lettuces were observed to be infected after 6 days. These results suggest that the nonpathogenic strain P. carotovorum subsp. carotovorum. Pcc21-M15 and E. coli (pRG3431) are effective to soft-rot disease suppression.