Development of PCR-Based Detection System for Soft Rot Pectobacteriaceae Pathogens Using Molecular Signatures

Pectobacterium and Dickeya species, usually referred to as soft rot Enterobacteriaceae, are phytopathogenic genera of bacteria that cause soft rot and blackleg diseases and are responsible for significant yield losses in many crops across the globe. Diagnosis of soft rot disease is difficult through visual disease symptoms. Pathogen detection and identification methods based on cultural and morphological identification are time-consuming and not always reliable. A polymerase chain reaction (PCR)-based detection method with the species-specific primers is fast and reliable for detecting soft rot pathogens. We have developed a specific and sensitive detection system for some species of soft rot Pectobacteriaceae pathogens in the Pectobacterium and Dickeya genera based on the use of species-specific primers to amplify unique genomic segments. The specificities of primers were verified by PCR analysis of genomic DNA from 14 strains of Pectobacterium, 8 strains of Dickeya, and 6 strains of non-soft rot bacteria. This PCR assay provides a quick, simple, powerful, and reliable method for detection of soft rot bacteria

Augmenting the Pressure-Based Pasteurization of Listeria monocytogenes by Synergism with Nisin and Mild Heat

The current study investigated Listeria monocytogenes inactivation using mild heat with elevated hydrostatic pressure and nisin under buffered condition. A four-strain pathogen mixture was exposed to 0 (control) and up to 9 min of (1) 4 °C elevated pressure; (2) 4 °C elevated pressure and nisin; (3) 4 °C nisin; (4) heat at 40 °C; (5) 40 °C elevated pressure; (6) 40 °C elevated pressure and nisin; and (7) 40 °C nisin. Elevated hydrostatic pressure at 400 MPa (Hub880 Explorer, Pressure BioScience Inc., Easton, MA, USA) and nisin concentration of 5000 IU/mL were used in the trials. Analyses of variance were conducted, followed by Dunnett’s- and Tukey-adjusted means separations. Under conditions of these experiments, nisin augmented (p \u3c 0.05) decontamination efficacy of 40 °C heat and elevated hydrostatic pressure treatments, particularly at treatment interval of 3 min. This synergism with nisin faded away (p ≥ 0.05) as the treatment time for thermal, high-pressure, and thermal-assisted pressure processing increased. The results of our study, thus, exhibit that practitioners and stakeholders of pressure-based technologies could benefit from synergism of mild heat and nisin for short-term, high-pressure pasteurization treatments to achieve microbial safety and economic feasibility comparable to traditional heat-treated products

Fate and Biofilm Formation of Wild-Type and Pressure-Stressed Pathogens of Public Health Concern in Surface Water and on Abiotic Surfaces

Since the historic outbreak near Broad Street in London, which serves as cornerstone of modern epidemiology, infectious diseases spread in surface and sub-surface water has been a persisting public health challenge. The current study investigated persistence of wild-type and pressure-stressed Listeria monocytogenes, Escherichia coli O157:H7, and non-typhoidal Salmonella enterica serovars in surface water stored aerobically for up to 28 days at 5, 25, and 37 °C. Additionally, biofilm formation of wild-type and pressure-stressed non-typhoidal Salmonella serovars were monitored on surface of stainless steel and rubber coupons for 28 days at 25 and 37 °C. While L. monocytogenes exhibited a lower (p \u3c 0.05) survival rate at 5 °C, relative to the two Gram-negative pathogens, at higher temperatures of 25 and 37 °C, all three pathogens exhibited similar (p ≥ 0.05) trends for survival in surface water. Both wild-type and pressure-stressed Salmonella serovars in the vast majority of tested times, temperatures, and surfaces exhibited comparable (p ≥ 0.05) persistence and biofilm formation capability. Our study thus indicates the occurrence of contamination could lead to prolonged survival of these microorganisms in low-nutrient environments and highlights the need for preventive measures such as those articulated under Produce Safety Rule of the U.S. Food Safety Modernization Act

The Bacterial Soft Rot Pathogens, Pectobacterium carotovorum and P. atrosepticum, Respond to Different Classes of Virulence-Inducing Host Chemical Signals

Soft rot bacteria of the Pectobacterium and Dickeya genera are Gram-negative phytopathogens that produce and secrete plant cell wall-degrading enzymes (PCWDE), the actions of which lead to rotting and decay of their hosts in the field and in storage. Host chemical signals are among the factors that induce the bacteria into extracellular enzyme production and virulence. A class of compounds (Class I) made up of intermediate products of cell wall (pectin) degradation induce exoenzyme synthesis through KdgR, a global negative regulator of exoenzyme production. While the KdgR− mutant of P. carotovorum is no longer inducible by Class I inducers, we demonstrated that exoenzyme production is induced in this strain in the presence of extracts from hosts including celery, potato, carrot, and tomato, suggesting that host plants contain another class of compounds (Class II inducers) different from the plant cell wall-degradative products that work through KdgR. The Class II inducers are thermostable, water-soluble, diffusible, and dialysable through 1 kDa molecular weight cut off pore size membranes, and could be a target for soft rot disease management strategies

Sensitivity of Planktonic Cells of Staphylococcus aureus to Elevated Hydrostatic Pressure as Affected by Mild Heat, Carvacrol, Nisin, and Caprylic Acid

Current study investigated effects of elevated hydrostatic pressure exposure in the presence of mild heat and natural antimicrobials against Staphylococcus aureus. Hydrostatic pressure of 350 to 550 MPa with nisin (5000 IU/mL), carvacrol, or caprylic acid (0.5% v/v) were applied for the reduction in four-strain mixture of S. aureus in HEPES buffer at 4 and 40 °C for up to 7 min. Results were statistically analyzed by ANOVA and D-values were additionally calculated using best-fitted linear model. Prior to exposure to treatments at 4 °C, counts of the pathogen were 7.95 ± 0.4 log CFU/mL and were reduced (p \u3c 0.05) to 6.44 ± 0.3 log CFU/mL after 7 min of treatment at 450 MPa. D-value associated with this treatment was 5.34 min (R2 = 0.72). At 40 °C, counts were 8.21 ± 0.7 and 5.77 ± 0.3 log CFU/mL before and after the 7-min treatments, respectively. D-value associated with 40 °C treatment was 3.30 min (R2 = 0.62). Application of the antimicrobials provided additional pathogen reduction augmentation for treatments \u3c 5 min. The results of the current study could be incorporated for meeting regulatory requirements such as Food Code, HACCP, and Preventive Control for Human Food of Food Safety Modernization Act for assuring microbiological safety of products against this prevalent pathogen of public health concern

Customer satisfaction in restaurant service : Naan-n-Curry, a Bangladeshi restaurant in Jakobstad

Customer satisfaction is an essential factor for any business organization in terms of deriving revenue, sales, growth and overall performance. Customer satisfaction ensures a strong spot for business companies with an increased competitive advantage for the companies in the market. Considering the issue, this thesis focuses on customer satisfaction and its importance by focusing on what the customers want and what the company should do to retain and attract customers. The author has taken Naan-n-Curry, which is a start-up restaurant trying to get established in the restaurant market of Jakobstad, Finland. The first four chapters of this thesis focused on the theoretical part in which the author tried to discuss about the commissioner, customer satisfaction and the impact and importance of customer satisfaction. Chapter four of this thesis shed light on multiple ways of achieving customer satisfaction. The objectives of the thesis were primarily two: the first one was to determine the factors that contribute to customer pleasure, and the second one was to determine how Naan-n-Curry uses these elements in combination with technological support to please its clients. The author discussed research methodologies in the second part of this thesis. A survey questionnaire consisting of 18 questions was prepared and a total of 50 responses were received. The survey data analysis and results have been presented in chapter six. The result suggests that, regular customers visit the restaurant on a daily or weekly basis. They visit the restaurant on a daily or weekly basis due to the quality of the food and services they receive. Office workers and students who frequently have lunch at Naan-n-Curry make up the majority of its customers

Enhancing the Detection Process, Prevention and Sustainable Management of Soilborne Diseases in Tennessee Nursery Production

Phytophthora and Rhizoctonia root rot diseases are the most important problems to nursery grown ornamentals. First objective of this study was to develop methodology for reliable and sensitive screening of irrigation water for the presence of Phytophthora spp. in the commercial nursery operations. Among three concentration methods, filtration was the most effective and sensitive method for detecting low level of zoospores in the water followed by baiting and centrifugation. Middle Tennessee nursery irrigation water samples were confirmed as Phytophthora positive (P. syringae, P. cryptogea, P. drechsleri and P. hydropathica) using baiting and filtration methods followed by culturing on PARPH-V8 medium, serological and molecular detection assays. Second objective of this study was to evaluate the efficacy of selected biocontrol products and fungicides against Phytophthora root rot of hydrangea cultivars and Rhizoctonia root rot of Viburnum odoratissimum in greenhouse and field experiments. Both greenhouse and field experiments showed that Segovis, Empress Intrinsic, Subdue Maxx, MBI110 and TerraClean 5.0 + TerraGrow program significantly reduced Phytophthora root rot severity; but Mural, Empress Intrinsic, Paegant Intrinsic and TerraClean 5.0 + TerraGrow program significantly reduced Rhizoctonia root rot severity compared to the non-treated inoculated control and other treatments. Third objective of this study was to evaluate the efficiency of soil solarization with and without organic inputs to control soilborne diseases. Soil solarization by itself and incorporated with selected biofumigant cover crops (mustard, mighty mustard, turnip, radish and astro arugula) good quality compost and mustard meal amendments were evaluated against soilborne pathogens under field conditions. On-farm experiments of solarization with or without incorporated biofumigant cover crops as well as mustard meal and compost amendments significantly reduced root rot diseases in Kwanzan flowering cherry cuttings compared to the non-treated, inoculated control. In TSUNRC field experiments, solarization with or without incorporated biofumigant cover crops as well as mustard meal and compost amendments significantly reduced Phytophthora root rot disease of boxwood rooted cuttings and Rhizoctonia root rot disease of V. odoratissimum compared to the non-treated, inoculated control plots. There were no significant differences among the treatments in reducing root rot disease severities in both on-farm and TSUNRC field experiments

Development of Genome-Directed, PCR-Based Molecular Diagnostic System for Soft Rot Bacteria

Pectobacterium spp and Dickeya spp are phytopathogenic bacteria that cause soft rot disease. Identification of soft rot pathogen is difficult with visual disease symptoms. This is especially true when different pathogen cause similar diseases in different plants. There are numerous soft rot pathogen detection methods based on culture and morphological identification but they are time-consuming, and not always reliable. A PCR-based diagnostic method with the species-specific primer pairs is fast, and reliable to detect soft rot pathogen. This study uses three pairs of species-specific primers to develop a diagnostic system for soft rot bacterial pathogens in the Pectobacterium and Dickeya species. The specificity of the primers was verified by PCR analysis of genomic DNA from 14 strains of Pectobacterium, 8 strains of Dickeya, and 6 strains of non-soft rot bacteria. This species-specific PCR assay provides quick, simple, powerful, and reliable detection of soft rot bacteria

EVALUATION OF MICROBIOLOGICAL SHELF LIFE AT DIFFERENT TEMPERATURES AND FATE OF ESCHERICHIA COLI O157:H7 INOCULATED IN PASTEURIZED MILK**.

According to a Centers for Disease Control and Prevention report, achieving safer and healthier foods had been one of the top 10 achievements of 20th century. Shiga-toxin-producing Escherichia coli O157:H7 is a well-known food-borne pathogen that can cause food borne diseases in humans. To control E. coli O157:H7 in foods, thermal process has been widely used, but there is a possibility to recover some sub – lethal microorganisms during storage period. Providing the additional assurance of thermal processing of food, some natural bioactive compounds can be the effective possible alternatives. There is not much information about the shelf life of food against E. coli O157:H7 at different temperatures has been reported. So, there is a need of some study to understand the fate and survival of E. coli O157:H7 during storage period. The current study investigated persistence of wild-type E. coli O157:H7 serovars in sterile milk stored aerobically for up to 28 days at 4°C and 37°C. Additionally, survival of E. coli O157:H7 against the synergistic effect of heat and caprylic acid in sterile milk were monitored after heat shock at 4°C and 37°C for 28 days. In this study 107 CFU/ml inoculum level and 0.5% caprylic acid were used. Inoculated samples were heat shocked with 63 ℃ for 30 minutes and 72 ℃ for 15 sec. Treatments at 63 ℃ for 30 minutes and the synergistic effect of caprylic acid with 63 ℃ for 30 minutes exhibited the similar trend for survival which were about 4.1 and 3.2 log CFU/ml reduction of E. coli O157:H7 respectively after 28 days at both 4°C and 37°C. Our study thus indicates the occurrence of contamination could lead to prolonged survival of these microorganisms in high and low-nutrient environments and highlights the need for preventive measures

Development of PCR-Based Detection System for Soft Rot Pectobacteriaceae Pathogens Using Molecular Signatures

Pectobacterium and Dickeya species, usually referred to as soft rot Enterobacteriaceae, are phytopathogenic genera of bacteria that cause soft rot and blackleg diseases and are responsible for significant yield losses in many crops across the globe. Diagnosis of soft rot disease is difficult through visual disease symptoms. Pathogen detection and identification methods based on cultural and morphological identification are time-consuming and not always reliable. A polymerase chain reaction (PCR)-based detection method with the species-specific primers is fast and reliable for detecting soft rot pathogens. We have developed a specific and sensitive detection system for some species of soft rot Pectobacteriaceae pathogens in the Pectobacterium and Dickeya genera based on the use of species-specific primers to amplify unique genomic segments. The specificities of primers were verified by PCR analysis of genomic DNA from 14 strains of Pectobacterium, 8 strains of Dickeya, and 6 strains of non-soft rot bacteria. This PCR assay provides a quick, simple, powerful, and reliable method for detection of soft rot bacteria