Organisms In Food Research Articles

Rapid Detection of Salmonella spp. in Foods by Combination of a New Selective Enrichment and a Sandwich ELISA Using Two Monoclonal Antibodies against Dulcitol 1-Phosphate Dehydrogenase

A rapid-detection method was developed for food-borne dulcitol-positive Salmonella spp. in foods that involves a new preenrichment and selective enrichment system and a sandwich ELISA using two monoclonal antibodies against dulcitol 1-phosphate dehydrogenase. Preenrichment and selective enrichment were in Enterobacteriaceae enrichment mannitol (EEM) broth at 42°C for 6 h and in a new dulcitol-magnesium chloride-pyridinesulfonic acid brilliant green-novobiocin (DMPBN) medium at 42°C for 27 h, respectively. The cells were collected from the selective enrichment culture and suspended in 0.1 ml of 1 N NaOH for 2 min. The solution was neutralized with 0.1 ml of 2M Tris-HCl buffer (pH 7.5) and the mixture was used as a sample for ELISA. The detection sensitivity of the ELISA was 105 CFU of Salmonella spp. per ml of culture. Competing non-Salmonella organisms in raw food did not interfere with the detection of Salmonella cells even when present at 107: 1 (non-Salmonella: Salmonella ratio) in food. Nonmotile Salmonella gallinarum was detected by the ELISA. The minimum detectable number of initial inoculum of Salmonella typhimurium was 0.69 CFU/25 g of raw chicken after the preenrichment in EEM broth and the selective enrichment in DMPBN medium. The present ELISA method required a total analysis time of 36 h including the preenrichment and selective enrichment periods. The ELISA method was compared with a conventional cultural method for the detection of Salmonella cells in 130 samples of raw foods. Of the samples tested, 16 were Salmonella-positive and 114 samples were negative by both methods. False-positive and false-negative results were not encountered.

Health risk assessment of Listeria monocytogenes in Canada

In this review, the major steps used in the formulation of a health risk assessment for Listeria monocytogenes in foods are discussed. Data is given on the numbers of human listeriosis cases reported in Canada along with the current Canadian regulatory policy on L. monocytogenes. Four major steps in the health risk assessment of this organism in foods, namely, hazard identification, hazard characterization, exposure assessment and risk characterization, were examined. For hazard characterization, since it is known that no direct human dose response data is available for L. monocytogenes, a flexible dose response model called the Weibull-Gamma model was evaluated. For the exposure assessment, pâté and soft cheese, both high-risk foods in terms of listeriosis infection, were used as prototypes in some of the models that were used. Using disappearance data for cheese and 100 g as a typical serving, the data suggested an average of 102 servings per capita, per year in Canada. As a rough approximation, for L. monocytogenes, reference ID 10 and ID 90 dose levels of response for both normal and high risk populations were given as 10 7 and 10 9 for normal individuals, and 10 5 and 10 7 for high-risk people. The corresponding dose response models were graphically displayed. These models exhibited a higher degree of susceptibility and less host/pathogen heterogeneity for the higher risk group. The range of doses between the ID 10 and ID 90 reference values corresponded roughly to levels associated with cases of listeriosis. In the risk characterization stage, dose response data was combined with some predictive.

PRODUCTION AND WESTERN BLOT CHARACTERIZATION OF MONOCLONAL ANTIBODIES SPECIFIC FOR CAMPYLOBACTER JEJUNI AND CAMPYLOBACTER COLI

Reference strains of the Lior serogroups of Campylobacter jejuni and C. coli most frequently encountered in human infections were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and western blotting. Seven components appeared to be common to all strains. Purified components were obtained from one strain by electrophoretic separation and elution from preparative SDS‐PAGE gels. The purified components were used to immunize BALB/c mice for monoclonal antibody production and to screen hybridoma tissue culture supernatants for specific antibody by enzyme immunoassay. Antibodies from selected hybridomas were characterized on western blots of C. jejuni, C. coli, C. fetus, Salmonella typhimurium, Proteus vulgaris, Citrobacter diversus and Escherichia coli whole cell lysates. At least nine of the monoclonal antibodies appeared to be specific for C. jejuni and C. coli and thus may be useful in tests for the rapid detection of these organisms in foods and other specimens.

Survival of E. coli and Salmonella after Chilling and Freezing in Liquid Media

ABSTRACTPredictive microbiology requires a sound basis of observed results, and factors that contribute to death of bacterial cells, before accurate equations can be developed. The effect of chilling and freezing strains of Escherichia coli and salmonella serotypes in nutrient broth, a noninhi‐bitory liquid medium, was investigated. The phase of growth, small changes in composition of test medium, and sub‐cultures made after primary isolation, influenced survival. Therefore, such influences must be considered when attempting to extrapolate results Erom pure cultures on laboratory media, to predict behavior of similar organisms in foods during chilling and freezing.

Metabolic and Structural Sites of Damage in Heat- and Sanitizer-Injured Populations of Listeria monocytogenes

Two food isolates of Listeria monocytogenes (strains ATCC 51414 and F5027) were sublethally injured by exposure to heat (56°C for 20 min) or to a chlorine sanitizer (Antibac, 100 ppm for 2 min). Percent injury following treatment ranged from 84% to 99%. Injured Listeria were repaired in Listeria repair broth (LRB) at 37°C. Comparison of the repair curves generated by each method indicated that the time for repair was greater for sanitizer-injured cells (14 h) than for heat-injured cells (5 h). Sites of injury were determined by repairing heat- and sanitizer-treated Listeria in LRB supplemented with one of the following inhibitors: rifampicin (10 and 20 μg/ml), chloramphenicol (5 μg/ml), cycloserine D (10 and 20 μg/ml), and carbonyl cyanide m-chlorophenyl-hydrazone(CCCP) (2.5 μg/ml). In both heat- and sanitizer-injured populations, a total inhibition of repair was seen following incubation with rifampicin, chloramphenicol and CCCP. These results clearly indicate a requirement for mRNA, protein synthesis, and oxidative phosphorylation for repair to occur. The cell wall is not a site of damage since cycloserine D had no effect on repair of heat- or sanitizer-injured Listeria. Investigation of damage to the cell membrane showed that stress caused by sublethal heat or sanitizer did not allow proteins or nucleotides to leak into the medium. The recognition of injury and repair in Listeria will lead to improved methods of detection and ultimately to control strategies which prevent outgrowth of this organism in foods.

Ability of Bdellovibrio bacteriovorus 109J to Lyse Gram-Negative Food-Borne Pathogenic and Spoilage Bacteria

Bdellovibrios are a group of aerobic, predatory bacteria which attack, penetrate and grow in many species of gram-negative bacteria, causing the lysis of the invaded prey organism. Bdellovibrio bacteriovorus strain 109J varied in its ability to lyse 32 bacterial strains comprising six genera of food-borne pathogens and spoilage organisms. The reduction in the levels of the prey bacteria ranged from 0.1 to 7.7 log-values after 7 h of incubation at 30°C. Escherichia coli strain 2239-69 (pathogenic serotype 026:H11) was lysed most effectively at temperatures between 30 and 37°C, however, lysis also occurred at 12 and 19°C when the incubation period was extended to 24 h. Bdellovibrio bacteriovorus was effective in reducing the level of E. coli 2239-69 at pH 5.6 to 8.6. Increasing the Bdellovibrio: E. coli ratio resulted in a more rapid E. coli reduction. This study demonstrated the potential usefulness of bdellovibrios for the biological control of pathogenic and spoilage organisms in foods.

The potential of lactic acid bacteria for the production of safe and wholesome food

By tradition lactic acid bacteria (LAB) are involved in the production of fermented foods. These constitute one quarter of our diet and are characterized by a safe history, certain beneficial health effects, and an extended shelf life when compared with raw materials. The various fermenting substrates are habitats for specific LAB that differ in their metabolic potential. The health effects exerted by LAB are the following: 1. Production of lactic acid and minor amounts of acetic and formic acid. These cause: a drop in pH and thereby growth inhibition of food spoiling or poisoning bacteria; killing of certain pathogens; detoxification by degradation of noxious compounds of plant origin (usually in combination with plant-derived enzymatic activities). 2. Production of antimicrobial compounds (e.g. bacteriocins, H2O2, fatty acids). 3. Probiotic effects as live organisms in food. The wholesomeness of LAB can also be extended to fields outside human nutrition, as they may act as probiotics in animal production or as plant protectives in agriculture and thus contribute to healthy raw materials for food production. Modern concepts or perspectives of the application of LAB include the following: 1. Selection of the best adapted and safely performing LAB strains. 2. Selection of strains with probiotic effects. 3. Selection of strains with health-promoting effects (e.g. production of vitamins or essential amino acids, anti-tumour activity). 4. Selection of strains with food protective activities (inhibiting spoilage or food pathogens). These strains can be added to food or used as starters in food fermentations. They may be found as wild-type organisms or can be obtained by genetic engineering.(ABSTRACT TRUNCATED AT 250 WORDS)

The Conditions Necessary for Sufficient Isolation of Causative Organisms in Food Poisoning Outbreaks

The Conditions Necessary for Sufficient Isolation of Causative Organisms in Food Poisoning Outbreaks

Current Research on Listeria monocytogenes in Foods: an Overview

Listeria monocytogenes is well-recognized as a foodborne pathogen. Two aspects of its control in foods are improvement in analytical methods and, secondly, the development of new methods to control its growth in foods. Much progress has been made since 1985 in developing both conventional and rapid methods for detecting L. monocytogenes in foods. When using conventional methodology it is readily acknowledged that the use of two methods, rather than one, will increase the recovery rates of the organism from naturally contaminated foods. Some of the newer rapid methods will be specifically targeting L. monocytogenes and not the entire Listeria genus as with previous kits. In the future many additional rapid test kits will come onto the market, as is the case now for Salmonella testing. The problem lies in proper evaluation of all of the kits. Advances have also been made in identification kits with at least two new tests showing great promise, the API™ Listeria, which can identify all species in the genus, and the Listeria ACCUPROBE™, which will specifically identify L. monocytogenes. Secondly, a lot of work has been done recently to develop new ways of controlling the growth of L. monocytogenes through the application of the “hurdle” concept. Research is concentrated in the area of biological controls by using starter cultures. Many different organisms along with their respective bacteriocins, have been found to have either bacteriostatic or bactericidal effects on the organism. Future work may concentrate on the use of genetically modified organisms that produce at least two different bacteriocins and on increasing the stability of pure bacteriocins in foods. The “hurdle” that still remains, however, is regulatory acceptance of the use of genetically modified organisms or pure bacteriocins in foods. We are still not much closer than we were 8 years ago to knowing the minimum infectious dose of the organism, and various countries have adopted different policies regarding the presence of L. monocytogenes in foods. It appears that more countries may be moving towards establishing tolerance levels for the presence of the organism in foods, especially for those low-risk foods which do not support growth of the organism and for those foods with a very short shelf life.

Cryptosporidium and Giardia as Agents of Foodborne Disease

Infections by the protozoan parasites of the genera Cryptosporidium and Giardia can be asymptomatic or cause gastroenteritis in immunocompetent people. However, in immunocompromised individuals, the infections can be more severe and even life threatening. Both parasites are common waterborne pathogens, but on occasion they may be foodborne or transmitted by body contact. In this review, several aspects of Cryptosporidium and Giardia are discussed including their life cycles, resistance to physical and chemical agents, routes of transmission to humans, the nature of the disease caused by the parasites, and detection of the organisms in water, feces, and food. Documented incidents in which Cryptosporidium or Giardia contaminated foods were implicated as cause of gastroenteritis are discussed to illustrate conditions leading to foodborne outbreaks and to suggest means of prevention and control of the parasites when present in foods.

RAPID METHODS AND AUTOMATION IN FOOD MICROBIOLOGY: BEYOND DELPHI FORECAST1

Routine microbiological analysis of foods has traditionally involved estimation of the total numbers of organisms by direct microscopy, viable counts or dye reduction, etc. The conventional methods have been fully or partially automated and alternative methods for estimation of microbial populations based on measurement of amplitude of selected parameter(s) have been introduced over the past two decades. The Delphi Technique is a method for technological forecasting helpful in planning future business activities, particularly in the areas of rapid technological development. In 1980–1981, a Delphi survey was conducted to develop a consensus view of future development in rapid and automated methods in food microbiology.The survey predicted that traditional methods for enumeration organisms in foods, i.e., colony count methods will be superseded by automated and mechanized methods for colony count by the end of the century. However, prediction of acceptance of alternative methodologies for detection and characterization of food‐borne pathogens, including the gene probes and the PCR based methods have been introduced. This presentation is designed to review these new technologies as a summary of this symposium.

New Molecular Methods for the Detection of Bacteria in Food

In order to maintain food safety standards, conventional microbiological methods are still being used to detect bacteria and other organisms in food. However, these techniques are not ideal, as often it can be many days before results are known‐which may be of particular economic importance for those foods with a short shelf‐life. The introduction of newer technology, such as nucleic acid probe and related amplification technology in other fields, has transformed the detection of many organisms. The Polymerase Chain Reaction (PCR) allows nucleic acid probes, with their inherent specificity, to be used to detect organisms present in very low numbers within a short period of time. However, at present, in food microbiology, there are technical problems with using the PCR, as certain components in food interfere with the reaction. When these problems are resolved and with prospects for semi‐automation of the PCR technique, there should be enormous potential for the rapid detection of bacteria in foods, with consequent benefits to the food industry and to consumers.

Experimental enzyme-linked amperometric immunosensors for the detection of salmonellas in foods.

Two enzyme-linked amperometric immunosensors specific for salmonellas were developed as rapid methods for quantifying and detecting these organisms in pure cultures and foods. Both used alkaline phosphatase as the enzyme reporter molecule but one system used phenyl phosphate as the substrate followed by the electrochemical detection of phenol at a polarized platinum electrode. The other system incorporated an enzyme amplification step and relied on the electrochemical detection of a reduced mediator, ferrocyanide. Both assays were rapid (4 h) and specific and generated salmonella-dependent signals above 10(4) cfu/ml (phenyl phosphate system) or 10(5) cfu/ml (enzyme amplified system) in pure cultures and samples of several foods, although the results with beef samples showed considerable variation. Both systems were able to detect low (1-5 cfu/g or /ml) numbers of salmonellas in foods after non-selective (18 h) and selective (22 h) enrichment steps but four samples, out of 147, gave false positive results. False positive results were eliminated by reducing the enrichment steps to 6 h and 18 h respectively (90 samples).

Microbiological Reference Values for Foods: A European Perspective

Microbiological criteria for food products and meals serve to gauge the results obtained upon monitoring samples from manufacturing plants or catering units which strictly adhere to good manufacturing practices (GMP). Hazardous practices thus already having been eliminated, the aim of monitoring is to detect and, above all, immediately correct accidental failures in processing or preparation. An essential element of this system of monitoring is that the number of criteria used is kept to a minimum by carefully selecting the criteria of major ecological significance. In view of the sporadic and erratic distribution of pathogenic organisms in foods from well-run manufacturing plants and food service operations, criteria for disease agents are used only occasionally. Where they are used, their selection and detection methodology require intensive and expert scrutiny, including (i) careful designation and precise definition of the relevant agents of disease, toxin formers, or preformed toxins; (ii) prescription of very carefully validated and standardized methods, usually including a resuscitation step; and (iii) mathematically correct interpretation of failure to detect particular infectious or toxinogenic organisms. On the other hand, "marker" organisms ("indicator" and "index" organisms, according to Ingram) are frequently used. These should also be carefully selected by ecological surveys on each specific food product; in particular, their value for reliably revealing deficiencies in hygiene or handling should be assessed. The applicability of the entire group of Enterobacteriaceae (or particular parts of that taxon), Enterococcus species, Staphylococcus aureus, and streptococci of the "mitis-salivarius" group are critically examined. Numerical reference values for microbiological criteria are derived by mathematical treatment from data obtained in surveys of products produced under GMP previously validated by safety analysis. Target values thus derived will include tolerance limits and policies recommended in case high results are obtained. Consequently, "3-class sampling plans" (according to ICMSF) are to be used in most instances.

Introduction

The use of legislation, scientific knowledge and education in controlling the proliferation of pathogenic organisms in food have been discussed. The need for refrigerants such as liquid nitrogen in maintaining the low temperatures required for chilled foods rather than ones which destroy the ozone layer or add to the greenhouse effect has been indicated.

16 Methods for Detecting Microbial Pathogens in Food and Water

Newly developed methods for the detection of bacteria and viruses have provided microbiologists with the means to rapidly identify and monitor specific microorganisms in food and water. Traditional methods of testing involve culture techniques to increase the numbers of the organism to a detectable level, followed by isolation and biochemical identification. This chapter focuses on the methodologies to detect pathogens and indicator organisms; however, the methods described are applicable to most bacteria. As detection and isolation methods have improved, a growing number of pathogens have been identified as important food- and waterborne pathogens. This chapter describes the use of nucleic acid and antibody probes that have the potential to circumvent the need to culture the organism prior to identification. Nucleic acid probes have become a valuable diagnostic reagent in the identification of human and animal pathogens and have made possible the identification of viruses and bacteria that are difficult, if not impossible, to cultivate. DNA probes have also proved to be a useful tool for identifying and monitoring the organisms in food and the environment.

Identification and enumeration of virulent Listeria strains

Recent outbreaks and a continuous increase in cases of listeriosis underscore the need for rapid, sensitive and reliable techniques to detect Listeria. Of the species of Listeria, only L. monocytogenes has been found to be associated with human infections. One factor which definitely contributes to its pathogenicity is the presence of hemolysins, although L. ivanovii and L. seeligeri also elaborate hemolysins. Based upon cloned hemolysin genes, we have developed DNA probes specifically for detecting L. monocytogenes. The technique combines growth of bacterial colonies on selective agar plates and DNA hybridization of these colonies on a solid matrix. This technique permits identification and enumeration, and the entire procedure can be completed in 3–4 days. Our method was found to be suitable for identifying and enumerating this organism in various foods, the main vehicle of human infection. Advantages and disadvantages of this technique are discussed and compared with other existing techniques.

FACTORS AFFECTING GROWTH OR SURVIVAL OF AEROMONAS HYDROPHILA IN FOODS

ABSTRACTRecent surveys of retail fresh foods (fish and seafood, poultry, red meat, raw milk and vegetables) have indicated the presence of organisms of the Aeromonas hydrophila group in virtually every sample examined. As part of these surveys, A. hydrophila was observed to increase in number during storage of the food at 5°C. Thus, other measures to control the growth of these organisms in retail fresh foods must be sought and evaluated; in addition, measures to destroy any A. hydrophila introduced into the foods must be investigated. This review discusses the various measures besides temperature (pH, NaCl) to control the growth of A. hydrophila in foods as well as their destruction by heat, irradiation, and sanitizers.

Simplified identification of aerobic spore-formers in the investigation of foods

Bacillus spp. are among the main spoilage organisms in food due to their versatile metabolism and heat-resistant spores. Except for B. cereus, no specific identification method is available, although information about Bacillus spp. would be useful in monitoring good manufacturing practice. A simplified identification scheme for aerobic spore-formers for routine laboratory use was elaborated by reducing the number of tests to be performed; 20 Bacillus spp. common in foods were considered. Besides cell morphology six biochemical characters were selected that proved most efficient in identification. In addition, methods were developed and modified to determine these characters by only 3 tests. By applying the simplified identification scheme, 766 strains of bacilli isolated from various foods were tested and species identity of 714 strains (93%) could be determined. Most aerobic spore-formers from foods were B. subtilis, B. pumilus and B. licheniformis. The spectrum of mesophilic bacilli isolated from natural sources was broad and comprised some 20 different species. Only one-third of the thermophilic isolates proved to be identical with, or similar to, B. stearothermophilus, while the rest were identified as five other species. The simplified method merits main application in ecological studies of foods.

Book Reviews

Book reviewed in this article:Current Topics in Microbiology and Immunology. Volume 127. THE WILD MOUSE I N IMMUNOLOGY (1986). Edited by M. Potter, J.H. Nadeau & M.P. Cancro.Introduction to Sterilization and Disinfection (1986). J.F. Gardner & M.M. Peel.Micro‐Organisms in Foods. Volume Sampling for Microbiological Analysis: Principles and Specific Applications (1986).