Research Note: Identification and characterization of Salmonella spp. in mechanically deboned chickens using pulsed-field gel electrophoresis.

Salmonella is one of the common foodborne bacteria, causing 80.3 million illnesses every year worldwide. This study was conducted to isolate and identify Salmonella enterica serovars from poultry samples responsible for causing foodborne poisoning in the Mississippi area, United States. A total of 55 S. enterica serovars-Enteritidis (6), Oranienburg (1), Schwarzengrund (8), Heidelberg (4), Kentucky (22), 4, [5], 12:i:- (1), Montevideo (2), Infantis (9), and multi serotypes (2)-were isolated from approximately 110 poultry samples. Through pulsed-field gel electrophoresis (PFGE) analysis, 8 to 13 bands were obtained. The profiles showed >90% similarity in strains within the same type. Consequently, PFGE could be a useful tool to determine chromosomal similarity (clonality of strains) that can be used to trace down epidemiologic sources and geographical origins of Salmonella.

Molecular characteristics, biofilm-forming abilities, and quorum sensing molecules in Vibrio parahaemolyticus strains isolated from marine and clinical environments in Korea.

Vibrio parahaemolyticus is an inhabitant of marine and estuarine environments and causes seafood-borne gastroenteritis in humans. In this study, an UltraFast LabChip Real-Time PCR assay was evaluated for rapid detection and quantification of pathogenic V. parahaemolyticus isolates. Escherichia coli and Vibrio harveyi were used as negative controls. Twenty-six tdh-positive, biofilm-producing V. parahaemolyticus isolates were analyzed by repetitive extragenic palindromic-polymerase chain reaction (REP-PCR). REP-PCR analysis showed that the majority of the V. parahaemolyticus isolates originated from seafood and that clinical specimens formed two major clusters at 92.8% and 32% similarity levels. The presence and quantification of Autoinducer-2 was carried out using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) after derivatization of Autoinducer-2 with 2, 3-diaminonaphthalene. The presence of tdh-positive V. parahaemolyticus in marine samples highlights the need for constant environmental monitoring to protect public health.

Variability in biofilm formation correlates with hydrophobicity and quorum sensing among Vibrio parahaemolyticus isolates from food contact surfaces and the distribution of the genes involved in biofilm formation.

Vibrio parahaemolyticus is one of the leading foodborne pathogens causing seafood contamination. Here, 22 V. parahaemolyticus strains were analyzed for biofilm formation to determine whether there is a correlation between biofilm formation and quorum sensing (QS), swimming motility, or hydrophobicity. The results indicate that the biofilm formation ability of V. parahaemolyticus is positively correlated with cell surface hydrophobicity, autoinducer (AI-2) production, and protease activity. Field emission scanning electron microscopy (FESEM) showed that strong-biofilm-forming strains established thick 3-D structures, whereas poor-biofilm-forming strains produced thin inconsistent biofilms. In addition, the distribution of the genes encoding pandemic clone factors, type VI secretion systems (T6SS), biofilm functions, and the type I pilus in the V. parahaemolyticus seafood isolates were examined. Biofilm-associated genes were present in almost all the strains, irrespective of other phenotypes. These results indicate that biofilm formation on/in seafood may constitute a major factor in the dissemination of V. parahaemolyticus and the ensuing diseases.

Optimization of hot water treatment for removing microbial colonies on fresh blueberry surface.

UNLABELLED: Blueberries for the frozen market are washed but this process sometimes is not effective or further contaminates the berries. This study was designed to optimize conditions for hot water treatment (temperature, time, and antimicrobial concentration) to remove biofilm and decrease microbial load on blueberries. Scanning electron microscopy (SEM) image showed a well-developed microbial biofilm on blueberries dipped in room temperature water. The biofilm consisted of yeast and bacterial cells attached to the berry surface in the form of microcolonies, which produced exopolymer substances between or upon the cells. Berry exposure to 75 and 90 °C showed little to no microorganisms on the blueberry surface; however, the sensory quality (wax/bloom) of berries at those temperatures was unacceptable. Response surface plots showed that increasing temperature was a significant factor on reduction of aerobic plate counts (APCs) and yeast/mold counts (YMCs) while adding Boxyl® did not have significant effect on APC. Overlaid contour plots showed that treatments of 65 to 70 °C for 10 to 15 s showed maximum reductions of 1.5 and 2.0 log CFU/g on APCs and YMCs, respectively; with acceptable level of bloom/wax score on fresh blueberries. This study showed that SEM, response surface, and overlaid contour plots proved successful in arriving at optima to reduce microbial counts while maintaining bloom/wax on the surface of the blueberries. PRACTICAL APPLICATION: Since chemical sanitizing treatments such as chlorine showed ineffectiveness to reduce microorganisms loaded on berry surface (Beuchat and others 2001, Sapers 2001), hot water treatment on fresh blueberries could maximize microbial reduction with acceptable quality of fresh blueberries.

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.

Proteome analysis of Azotobacter vinelandii ∆arrF mutant that overproduces poly-ß-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-ß-hydroxybutyrate (PHB). Proteins differentially expressed in the mutant were identified by gel-based proteomics and confirmed by real-time RT-PCR. 6-Phosphogluconolactonase and E(1) 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.

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.

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.

Overproduction of poly-beta-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-beta-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 approximately 61-, 18-, and eightfold, respectively, in the mutant. The phbR transcript of the activator PhbR for this operon was also approximately 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.