Yean-Sung Jung

Prevalence and contamination patterns of Listeria monocytogenes in catfish processing environment and fresh fillets.

Catfish skins, intestines, fresh fillets, processing surfaces at different production stages, chiller water and non-food contact surfaces were sampled for Listeria monocytogenes and other Listeria species. Among 315 samples, prevalence of L. monocytogenes, Listeria innocua and a group of Listeria seeligeri-Listeria welshimeri-Listeria ivanovii was 21.6, 13.0 and 29.5%, respectively. No Listeria grayi was detected in this survey. While no L. monocytogenes strains were isolated from catfish skins and intestines, the strains were found with a frequency of 76.7% in chilled fresh catfish fillets and 43.3% in unchilled fillets. L. monocytogenes and Listeria spp. were also detected in fish contact surfaces such as deheading machine, trimming board, chiller water, conveyor belts at different stages, and fillet weighing table. Among L. monocytogenes, 1/2b (47.0%), 3b (16.0%) and 4c (14%) were the predominant serotypes isolated, whereas 4b, 4e, 1/2c and 1/2a were detected at much lower frequencies. Genotype analyses of L. monocytogenes isolates using serotyping, pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed that chiller water represented an important contamination source of L. monocytogenes in the chilled catfish fillets of two processing facilities, whereas fillet weighing table significantly contributed to the catfish fillet contamination of the third facility. This study suggests that L. monocytogenes contamination in the processed catfish fillets originates from the processing environment, rather than directly from catfish. Results from this study can aid the catfish industry to develop a plant-specific proper cleaning and sanitation procedure for equipment and the processing environment designed to specifically target L. monocytogenes contamination.

Antibiotic resistance in Listeria species isolated from catfish fillets and processing environment.

Aims:  To investigate the susceptibility of 221 Listeria spp. (86 Listeria monocytogenes, 41 Listeria innocua and 94 Listeria seeligeri‐Listeria welshimeri‐Listeria ivanovii) isolated from catfish fillets and processing environment to 15 antibiotics.

Enhanced antimicrobial activity of starch-based film impregnated with thermally processed tannic acid, a strong antioxidant.

Starch-based films impregnated with fresh tannic acid (FTA/starch film) and thermally processed tannic acid (PTA/starch film) were assessed for inhibition of Escherichia coli O157:H7 and Listeria monocytogenes. Disc-diffusion assay revealed that the PTA/starch film showed larger clear zone around the film on the bacterial lawn than the FTA/starch film at the same tannic acid concentrations (0.45 to 4.5mg per disc). Viable cell count assays in tryptic soy broth showed that the PTA/starch film also had a stronger antimicrobial activity on these foodborne pathogens than the FTA/starch film. L. monocytogenes did not replicate in trypic soy broth containing the FTA/starch film for the first 8h but multiplied up to 9.22 log CFU/ml at 48 h of incubation. The PTA/starch film caused a 2.72-log decrease in L. monocytogenes cells over the same time period. While 5-log E. coli O157:H7 cells were inactivated by the FTA/starch film within 48 h, more than 7-log E. coli O157:H7 cells were killed by the PTA/starch film over the same period. The antimicrobial activity of FTA/starch and PTA/starch film was primarily pH independent. HPLC measurement of the FTA or PTA release from starch film in water revealed that their release kinetic curves were in well match with their inactivation curves for E. coli O157:H7 and L. monocytogenes in 0.1% peptone water. In addition to antimicrobial activity, FTA showed antioxidant activity on soybean oil by doubling the induction time of oil oxidation. PTA further enhanced the oxidative stability of the oil by 17%. These results suggested that the use of processed tannic acid in starch films could improve the safety and quality of foods.

Antimicrobial Effect of Water-Soluble Muscadine Seed Extracts on Escherichia coli O157:H7

Water-soluble extracts were prepared from purple (cultivar Ison) and bronze (cultivar Carlos) muscadine seeds with or without heating. The Ison extracts had strong antimicrobial activity against a cocktail of three strains of Escherichia coli O157: H7. This extract had higher acidity (pH 3.39 to 3.43), total phenolics (2.21 to 3.49 mg/ml), tartaric acid (5.6 to 10.7 mg/ml), tannic acid (5.7 to 8.1 mg/ml), and gallic acid (0.33 to 0.59 mg/ml) than did the Carlos extracts. Heat treatment on both extracts increased antimicrobial activity, possibly because of increased acidity, tartaric acid, total phenolics, and individual phenolics. Heating of Ison extracts increased ellagic acid up to 83%. Up to 10.7 mg/ml tartaric acid alone was not as effective against E. coli O157:H7 as were water-soluble seed extracts. This finding suggests the involvement of other factors, such as tannic and gallic acids, in inactivation of this pathogen. Water-soluble muscadine seed extracts may be useful for incorporation into juice and other beverage products as natural preservatives.

Small RNA ArrF Regulates the Expression of sodB and feSII Genes in Azotobacter vinelandii

Azotobacter vinelandii contains a prrF-like sequence in a noncoding region of the chromosome. Like the Pseudomonas aeruginosa PrrF small RNA-encoding genes, the expression of the sequence, herein named arrF (Azotobacter regulatory RNA involving Fe), was increased 100-fold in wild-type cells in response to iron depletion. The level of ArrF was also increased to the same degree in the iron-replete fur mutant, but down back to a wild-type level when this fur mutant was complemented with the wild-type fur gene. These results, with the location of arrF gene in a noncoding region, suggest that this gene encodes an iron-responsive small RNA whose expression is negatively regulated by the Fur–Fe2+ complex. Disruption of this arrF gene upregulated the expression of iron-containing superoxide dismutase and FeSII protein, whereas fur mutation or iron depletion decreased the level of their transcript. A short region in the 5′-untranslated region of each transcript was predicted to be quite complementary to the core sequence of ArrF, assuming that ArrF represses the expression of the genes under Fur control by an antisense RNA mechanism. However, unlike the P. aeruginosa PrrF that has extensive targets in the tricarboxylic acid cycle and glyoxylate cycle, ArrF had little effect on those genes. The findings that there is a poor overlap between ArrF and PrrF targets and that the FeSII gene, which is present only in the chromosome of nitrogen-fixing bacterial species, is controlled by ArrF suggest that ArrF might have unique targets, some of which are involved in N2 fixation.

Identification of Natural Antimicrobial Substances in Red Muscadine Juice against Cronobacter sakazakii

Red muscadine (Vitis rotundifolia Michx.) juices with natural organic, phenolic acids and polyphenol compounds were tested against Cronobacter sakazakii. The concentration of total phenolic compounds of commercial baby juices ranged from 176.7 to 347.7 mg/mL. Commercial baby juices showed poor antimicrobial activity, reducing less than 1-log of C. sakazakii in juice samples for 2 h at 37 degrees C. Red muscadine juices, regardless of processing methods (filtration, pasteurization, and sterilization), achieved a 6-log reduction of C. sakazakii in the same time period (2 h). The mixture of synthetic organic acids (malic and tartaric acids) and polyphenolic acid (tannic acid) showed strong antimicrobial activity against C. sakazakii. Among synthetic organic acids, tannic acid was undetected in commercial baby juices. Tannic acid showed the highest antimicrobial activity (1.4- to 3.8-log reduction) against C. sakazakii, while malic and tartaric acids showed less than 0.5-log reduction. These results suggest that red muscadine juice could be utilized as a natural antimicrobial in baby food formulations to inhibit C. sakazakii.

Antibacterial activity of fresh and processed red muscadine juice and the role of their polar compounds on Escherichia coli O157:H7

Aims:  The objectives of this research were to show the anti‐Escherichia coli O157:H7 effect of fresh (FRMJ) and processed red muscadine (Vitis rotundifolia) juice (PRMJ) and to discern the active compounds responsible for anti‐E. coli O157:H7.

Incidence and Persistence of Listeria monocytogenes in the Catfish Processing Environment and Fresh Fillets

Incidence of Listeria spp. in whole raw catfish, catfish fillets, and processing environments from two catfish processing facilities was determined in August 2008 and August 2009. Thirty-nine (18.4%) of 212 samples collected in August 2008 were positive for Listeria monocytogenes. Prevalences of Listeria species L. innocua and L. seeligeri-L. welshimeri-L. ivanovii were 11.3 and 23.6%, respectively. Of 209 samples collected in August 2009, 12.4% were positive for L. monocytogenes, 11% for L. innocua, and 19.6% for L. seeligeri-L. welshimeri-L. ivanovii. No Listeria grayi was detected in any of the samples. L. monocytogenes was not found in catfish skins and intestines, but was detected in catfish fillets, on food contact surfaces, and on non-food contact surfaces with frequencies of 45.0, 12.0, and 11.1%, respectively. In August 2008 isolates, serotypes 1/2b (62.2%) and 3b (15.6%) were frequently isolated, whereas the majority of the August 2009 isolates (92.3%) were serotype 1/2b. Genotyping analyses revealed that some genotypes of L. monocytogenes isolates were detected in one facility even after a year, but no persistence of L. monocytogenes was observed in the other facility. In addition, some L. monocytogenes isolates from fresh fillets showed genotypes that were either identical, or more than 90% similar, to those of L. monocytogenes isolates from food contact surfaces in the processing lines. The results of this study suggest that processing environment rather than whole raw catfish is an important source of L. monocytogenes contamination in the catfish fillets. These results should assist the catfish industry to develop better control and prevention strategies for L. monocytogenes.

Overproduction of poly-β-hydroxybutyrate in the Azotobacter vinelandii mutant that does not express small RNA ArrF

Azotobacter vinelandii contains an iron-regulatory small RNA ArrF whose expression is dependent upon the levels of iron and ferric uptake regulator. The deletion of this ArrF-encoding gene resulted in a 300-fold increase in the production of poly-β-hydroxybutyrate (PHB), a polymer of industrial importance. This ∆arrF mutant exhibited wild-type growth and growth-associated PHB production. Limited iron and aeration elevated the PHB production in the mutant as well as wild type. Real-time RT-PCR revealed that phbB, phbA, and phbC were upregulated ∼61-, 18-, and eightfold, respectively, in the mutant. The phbR transcript of the activator PhbR for this operon was also ∼11 times more abundant. The analysis of phbR transcript predicted a region of complementarity near its Shine–Dalgarno sequence that could potentially basepair with the conserved region of ArrF. These results suggest that ArrF represses the expression of PhbR in an antisense manner and derepression of this activator in the mutant elevates the expression of phbB, phbA, and phbC, resulting in the PHB overproduction.

Proteome analysis of Azotobacter vinelandii ∆arrF mutant that overproduces poly-β-hydroxybutyrate polymer

Azotobacter vinelandii ArrF is an iron-responsive small RNA that is under negative control of Ferric uptake regulator protein. A. vinelandii ∆arrF mutant that had a deletion of the entire arrF gene was known to overproduce poly-β-hydroxybutyrate (PHB). Proteins differentially expressed in the mutant were identified by gel-based proteomics and confirmed by real-time RT-PCR. 6-Phosphogluconolactonase and E1 component of pyruvate dehydrogenase complex, which leads to the production of NADPH and acetyl-CoA, were upregulated, while proteins in the tricarboxylic acid cycle that consumes acetyl-CoA were downregulated. Heat-shock proteins such as HSP20 and GroEL were highly overexpressed in the mutant. Antioxidant proteins such as Fe-containing superoxide dismutase (FeSOD), a putative oxidoreductase, alkyl hydroperoxide reductase, flavorprotein WrbA, and cysteine synthase were also overexpressed in the ∆arrF mutant, indicating that the PHB accumulation is stressful to the cells. Upregulated in the ∆arrF mutant were acetyl-CoA carboxylase, flagellin, and adenylate kinase, though the reasons for their overexpression are unclear. Among genes upregulated in the mutant, sodB coding for FeSOD and phbF encoding PHB synthesis regulator PhbF were negatively regulated by small RNA ArrF probably in an antisense mechanism. The deletion of arrF gene, therefore, would increase PhbF and FeSOD levels, which favors PHB synthesis in the mutant. On the other hand, glutamate synthetase, elongation factor-Tu, iron ABC transporter, and major outer membrane porin OprF were downregulated in the ∆arrF mutant. Based on the results, it is concluded that multiple factors including the direct effect of small RNA ArrF might be responsible for the PHB overproduction in the mutant.