Accurate identification of desired clones from 16S-23S rDNA spacer libraries using single PCR.
Explore the source record for details and available documents.
Base Sequence↗
Biomedical subjects
Lee-Ann Jaykus
Publications and source records attributed to Lee-Ann Jaykus.
A field study of the microbiological quality of fresh produce of domestic and Mexican origin.
Produce is responsible for an increasingly larger proportion of foodborne disease outbreaks. In particular, the globalization of the food supply may introduce new food safety risks and allow widespread distribution of contaminated food, particularly produce. The objectives of this study were to: (i) compare the overall quality of domestic and Mexican produce throughout the packing process; (ii) examine changes in microbiological quality of both domestic and Mexican produce at each stage of production and processing; and (iii) evaluate the prevalence of select pathogens on fresh produce, including leafy green, herbs, melons, and vegetables. Furthermore, we also sought to characterize the antibiotic resistance profiles of Enterococcus faecium and Enterococcus faecalis strains isolated from fresh produce. A total of 466 produce and matching environmental swab samples was collected from various locations in packing sheds in the southern US from November 2002 through December 2003. These samples were assayed by enumerative tests for total aerobic bacteria (APC), total coliforms, total Enterococcus, and E. coli. Produce samples were also analyzed for the presence of Salmonella, Listeria monocytogenes, Shigella, and E. coli O157:H7. A total of 112 E. faecium and E. faecalis isolates were further screened for antibiotic resistance using a panel of seventeen antibiotics. Overall, the microbiological quality of fresh produce ranged from 4.0 to 7.9 log(10) CFU/g (APC); less than 1.0 log(10) to 4.5 log(10) CFU/g (coliforms); less than 1.0 log(10) to 4.0 log(10) CFU/g (E. coli); and less than 1.0 log(10) to 5.4 log(10) CFU/g (Enterococcus). No Salmonella, Shigella, or E. coli O157:H7 were detected from the 466 25-g produce samples tested. However, three domestic cabbage samples were found to be positive for L. monocytogenes. Of the Enterococcus isolates, E. faecium had a higher degree of resistance to antibiotics in general, while Enterococcus spp. isolated from Mexican produce had a higher degree of antibiotic resistance when compared to strains isolated from produce samples of domestic origin. Despite increased attention to the role of imported produce in foodborne disease, this study does not support the assumption that domestic produce is of higher microbial quality than Mexican produce.
Anti-Bacterial Agents↗
Evaluation of BBL CHROMagar Listeria agar for the isolation and identification of Listeria monocytogenes from food and environmental samples.
The performance of BBL CHROMagar Listeria chromogenic agar for the detection of Listeria monocytogenes was evaluated for its ability to isolate and identify L. monocytogenes from food and environmental samples. The medium was compared to non-chromogenic selective agars commonly used for Listeria isolation: Oxford, Modified Oxford, and PALCAM. BBL CHROMagar Listeria had a sensitivity of 99% and 100% for the detection of L. monocytogenes from 200 natural and artificially inoculated food samples, respectively, with a colony confirmation rate of 100%. The sensitivity of non-chromogenic selective media for the detection of L. monocytogenes from these same samples was 97-99% with colony confirmation rates of 65-67.5%. From 93 environmental samples, BBL CHROMagar Listeria agar results correlated 100% with a Listeria spp. visual immunoassay (TECRA) performed on these same samples and the USDA-FSIS standard culture method for the isolation of L. monocytogenes. From environmental samples, the L. monocytogenes confirmation rate was 100% for BBL CHROMagar Listeria as compared to 50% for conventional agars tested. On BBL CHROMagar Listeria, L. monocytogenes forms a translucent white precipitation zone (halo) surrounding blue-pigmented colonies of 2-3 mm in diameter, with an entire border. BBL CHROMagar Listeria offers a high degree of specificity for the confirmation of suspect L. monocytogenes colonies, whereas non-chromogenic selective agars evaluated were not differential for L. monocytogenes from other Listeria species.
Agar↗
Consumer phase risk assessment for Listeria monocytogenes in deli meats.
The foodborne disease risk associated with the pathogen Listeria monocytogenes has been the subject of recent efforts in quantitative microbial risk assessment. Building upon one of these efforts undertaken jointly by the U.S. Food and Drug Administration and the U.S. Department of Agriculture (USDA), the purpose of this work was to expand on the consumer phase of the risk assessment to focus on handling practices in the home. One-dimensional Monte Carlo simulation was used to model variability in growth and cross-contamination of L. monocytogenes during food storage and preparation of deli meats. Simulations approximated that 0.3% of the servings were contaminated with >10(4) CFU/g of L. monocytogenes at the time of consumption. The estimated mean risk associated with the consumption of deli meats for the intermediate-age population was approximately 7 deaths per 10(11) servings. Food handling in homes increased the estimated mean mortality by 10(6)-fold. Of all the home food-handling practices modeled, inadequate storage, particularly refrigeration temperatures, provided the greatest contribution to increased risk. The impact of cross-contamination in the home was considerably less. Adherence to USDA Food Safety and Inspection Service recommendations for consumer handling of ready-to-eat foods substantially reduces the risk of listeriosis.
Animals↗
Consumer-phase Salmonella enterica serovar enteritidis risk assessment for egg-containing food products.
We describe a one-dimensional probabilistic model of the role of domestic food handling behaviors on salmonellosis risk associated with the consumption of eggs and egg-containing foods. Six categories of egg-containing foods were defined based on the amount of egg contained in the food, whether eggs are pooled, and the degree of cooking practiced by consumers. We used bootstrap simulation to quantify uncertainty in risk estimates due to sampling error, and sensitivity analysis to identify key sources of variability and uncertainty in the model. Because of typical model characteristics such as nonlinearity, interaction between inputs, thresholds, and saturation points, Sobol's method, a novel sensitivity analysis approach, was used to identify key sources of variability. Based on the mean probability of illness, examples of foods from the food categories ranked from most to least risk of illness were: (1) home-made salad dressings/ice cream; (2) fried eggs/boiled eggs; (3) omelettes; and (4) baked foods/breads. For food categories that may include uncooked eggs (e.g., home-made salad dressings/ice cream), consumer handling conditions such as storage time and temperature after food preparation were the key sources of variability. In contrast, for food categories associated with undercooked eggs (e.g., fried/soft-boiled eggs), the initial level of Salmonella contamination and the log10 reduction due to cooking were the key sources of variability. Important sources of uncertainty varied with both the risk percentile and the food category under consideration. This work adds to previous risk assessments focused on egg production and storage practices, and provides a science-based approach to inform consumer risk communications regarding safe egg handling practices.
Bacteriophage Typing↗
Surrogates for the study of norovirus stability and inactivation in the environment: aA comparison of murine norovirus and feline calicivirus.
Human noroviruses (NoVs) are the leading cause of food- and waterborne outbreaks of acute nonbacterial gastroenteritis worldwide. As a result of the lack of a mammalian cell culture model for these viruses, studies on persistence, inactivation, and transmission have been limited to cultivable viruses, including feline calicivirus (FCV). Recently, reports of the successful cell culture of murine norovirus 1 (MNV-1) have provided investigators with an alternative surrogate for human NoVs. In this study, we compared the inactivation profiles of MNV-1 to FCV in an effort to establish the relevance of MNV-1 as a surrogate virus. Specifically, we evaluated (i) stability upon exposure to pH extremes; (ii) stability upon exposure to organic solvents; (iii) thermal inactivation; and (iv) surface persistence under wet and dry conditions. MNV-1 was stable across the entire pH range tested (pH 2 to 10) with less than 1 log reduction in infectivity at pH 2, whereas FCV was inactivated rapidly at pH values < 3 and > 9. FCV was more stable than MNV-1 at 56 degrees C, but both viruses exhibited similar inactivation at 63 and 72 degrees C. Long-term persistence of both viruses suspended in a fecal matrix and inoculated onto stainless steel coupons were similar at 4 degrees C, but at room temperature in solution, MNV-1 was more stable than FCV. The genetic relatedness of MNV-1 to human NoVs combined with its ability to survive under gastric pH levels makes this virus a promising and relevant surrogate for studying environmental survival of human NoVs.
Animals↗
Application of classification and regression trees for sensitivity analysis of the Escherichia coli O157:H7 food safety process risk model.
Microbial food safety process risk models are simplifications of the real world that help risk managers in their efforts to mitigate food safety risks. An important tool in these risk assessment endeavors is sensitivity analysis, a systematic method used to quantify the effect of changes in input variables on model outputs. In this study, a novel sensitivity analysis method called classification and regression trees was applied to safety risk assessment with the use of portions of the Slaughter Module and Preparation Module of the E. coli O157:H7 microbial food safety process risk as an example. Specifically, the classification and regression trees sensitivity analysis method was evaluated on the basis of its ability to address typical characteristics of microbial food safety process risk models such as nonlinearities, interaction, thresholds, and categorical inputs. Moreover, this method was evaluated with respect to identification of high exposure scenarios and corresponding key inputs and critical limits. The results from the classification and regression trees analysis applied to the Slaughter Module confirmed that the process of chilling carcasses is a critical control point. The method identified a cutoff value of a 2.2-log increase in the number of organisms during chilling as a critical value above which high levels of contamination would be expected. When classification and regression trees analysis was applied to the cooking effects part of the Preparation Module, cooking temperature was found to be the most sensitive input, with precooking treatment (i.e., raw product storage conditions) ranked second in importance. This case study demonstrates the capabilities of classification and regression trees analysis as an alternative to other statistically based sensitivity analysis methods, and one that can readily address specific characteristics that are common in microbial food safety process risk models.
Consumer Product Safety↗
Consumer knowledge, storage, and handling practices regarding Listeria in frankfurters and deli meats: results of a Web-based survey.
Proper storage and handling of refrigerated ready-to-eat foods can help reduce the risk of listeriosis. A national Web-based survey was conducted to measure consumer awareness and knowledge of Listeria and to estimate the prevalence of the U.S. Department of Agriculture-recommended consumer storage and handling practices for frankfurters and deli meats. The demographic characteristics of consumers who are unaware of Listeria and who do not follow the recommended storage guidelines were also assessed. In addition, predictive models were developed to determine which consumers engage in risky storage practices. Less than half of the consumers surveyed were aware of Listeria, and most of those aware were unable to identify associated food vehicles. Awareness was lower among adults 60 years of age and older, an at-risk population for listeriosis, and individuals with relatively less education and lower incomes. Most households safely stored and prepared frankfurters. Most households stored unopened packages of vacuum-packed deli meats in the refrigerator within the U.S. Department of Agriculture-recommended storage guidelines (< or =14 days); however, many stored opened packages of vacuum-packed deli meats and freshly sliced deli meats for longer than the recommended time (< or =5 days). Men, more-educated individuals, and individuals living in metropolitan areas were more likely to engage in risky storage practices. This study identified the need to develop targeted educational initiatives on listeriosis prevention.
Adolescent↗
Upstream sample processing facilitates PCR detection of Listeria monocytogenes in mayonnaise-based ready-to-eat (RTE) salads.
Sample pretreatment to reduce volume and concentrate cells of the target organism(s) prior to molecular detection offers a useful supplement or alternative to cultural enrichment. The purpose of this study was to develop an upstream processing method to facilitate the detection of Listeria monocytogenes in ready-to-eat (RTE) salads by PCR. Potato salad, a model RTE commodity, was seeded with L. monocytogenes and processed by two alternative upstream sample processing methods (designated one-step and two-step centrifugation), followed by DNA extraction, PCR amplification, and Southern hybridization. The two-step method resulted in 1,000-fold improvements in the PCR detection limit, from 10(6) Cfu/g (no sample processing) to 10(3) Cfu/g. The two-step method was applied for upstream sample processing of four representative deli salad items artificially inoculated with L. monocytogenes at levels ranging from 10(1)-10(6) Cfu/g. Following DNA extraction, PCR amplification, and Southern hybridization, detection was achieved at input levels of 10(5) Cfu/g for chicken salad, 10(4) Cfu/g for macaroni salad, and 10(3) Cfu/g for potato and seafood salads. The two-step method reported here facilitates the production of a final sample concentrate of reduced volume and improved purity which was compatible with PCR amplification. This approach offers further progress in our efforts to reduce or eliminate cultural enrichment in an effort to speed time to results when applying molecular methods to the detection of pathogens in foods.
Blotting, Southern↗
A simple method for the direct detection of Salmonella and Escherichia coli O157:H7 from raw alfalfa sprouts and spent irrigation water using PCR.
The U.S. Food and Drug Administration recognizes that raw seed sprouts are an important cause of foodborne disease and is now recommending that either spent irrigation water or final product be screened for Salmonella and Escherichia coli O157:H7 as a means of assuring the safety of product intended for consumption. In an effort to streamline such testing efforts, a simple method to preconcentrate pathogens from sprouts and spent irrigation water was investigated to facilitate the direct (without prior cultural enrichment) detection of pathogens using the PCR technique. Alfalfa sprouts and spent irrigation water were seeded with Salmonella enterica serovar Typhimurium and E. coli O157:H7 at 10(-1) to 106 CFU/g or CFU/ml, respectively. Samples were blended (sprouts only) and then centrifuged at high speed to sediment the total bacterial population. The precipitate was processed for DNA isolation, PCR amplification, and amplicon confirmation by Southern hybridization. Mean pathogen recoveries after centrifugation ranged from 96 to 99% for both pathogens in both matrices. Using primers targeting the invA gene for Salmonella Typhimurium and the stx genes of E. coli O157:H7, it was possible to detect both pathogens in alfalfa sprouts at seeding concentrations as low as 10 CFU/g. PCR detection limits for both pathogens from spent irrigation water were 10(-1) CFU/ml, the equivalent of 100 CFU/liter of water. Because spent irrigation water is constitutionally simple, it is particularly well suited for bacterial concentration by simple centrifugation steps. In this study, progress was made toward development of a rapid, inexpensive, and sensitive method for the detection of sprout-associated pathogens that is relevant to current industrial practices and needs.
Colony Count, Microbial↗
Effect of food matrix and cell growth on PCR-based detection of Escherichia coli O157:H7 in ground beef.
The purpose of this work was (i) to investigate the feasibility of a previously reported upstream processing method for PCR template preparation to facilitate the detection of Escherichia coli O157:H7 from ground beef and (ii) to assess the impact of cell growth (no growth in the matrix versus growth in the matrix) on molecular detection limits. Two food matrices (autoclaved and raw ground beef) were evaluated in all studies. For no-growth experiments, 10-g meat samples were inoculated with 10(2) to 10(7) CFU/g E. coli O157:H7 and then homogenized. The homogenates were processed to remove large particulates and inhibitors using a two-phase upstream processing method consisting of two sequential centrifugation steps, the second of which used titanous hydroxide to facilitate bacterial immobilization. After upstream processing, sample concentrates were extracted for DNA isolation and amplified by PCR. For growth experiments, 10-g meat samples were inoculated at 1 CFU of E. coli O157:H7 per gram, allowed to grow to 10(2) to 10(7) CFU/g, and then processed for PCR assay. Cell recoveries after upstream processing ranged from 15.9 to 77.6% and were not facilitated by the use of titanous hydroxide, as compared with a saline control (P > 0.05). Bacterial cell recovery and PCR detection limits were similar when comparing autoclaved ground beef and raw ground beef, but cell recoveries were highly variable for raw ground beef samples in which E. coli O157:H7 cells were allowed to grow before processing for detection. Overall, PCR detection limits approximated 10(3) CFU/g of ground beef for all treatments. These results indicate that use of model food systems may not always provide an accurate replication of real-world conditions when evaluating PCR detection limits.
Animals↗
A field study of the microbiological quality of fresh produce.
The Centers for Disease Control and Prevention has reported that foodborne disease outbreaks associated with fruits and vegetables increased during the past decade. This study was conducted to characterize the routes of microbial contamination in produce and to identify areas of potential contamination from production through postharvest handling. We report here the levels of bacterial indicator organisms and the prevalence of selected pathogens in produce samples collected from the southern United States. A total of 398 produce samples (leafy greens, herbs, and cantaloupe) were collected through production and the packing shed and assayed by enumerative tests for total aerobic bacteria, total coliforms, total Enterococcus, and Escherichia coli. These samples also were analyzed for Salmonella, Listeria monocytogenes, and E. coli O157:H7. Microbiological methods were based on methods recommended by the U.S. Food and Drug Administration. For all leafy greens and herbs, geometric mean indicator levels ranged from 4.5 to 6.2 log CFU/g (aerobic plate count); less than 1 to 4.3 log CFU/g (coliforms and Enterococcus); and less than 1 to 1.5 log CFU/g (E. coli). In many cases, indicator levels remained relatively constant throughout the packing shed, particularly for mustard greens. However, for cilantro and parsley, total coliform levels increased during the packing process. For cantaloupe, microbial levels significantly increased from field through packing, with ranges of 6.4 to 7.0 log CFU/g (aerobic plate count); 2.1 to 4.3 log CFU/g (coliforms); 3.5 to 5.2 log CFU/g (Enterococcus); and less than 1 to 2.5 log CFU/g (E. coli). The prevalence of pathogens for all samples was 0, 0, and 0.7% (3 of 398) for L. monocytogenes, E. coli O157:H7, and Salmonella, respectively. This study demonstrates that each step from production to consumption may affect the microbial load of produce and reinforces government recommendations for ensuring a high-quality product.
Bacteria↗
Bacterial separation and concentration from complex sample matrices: a review.
The use of many rapid detection technologies could be expanded if the bacteria were separated, concentrated, and purified from the sample matrix before detection. Specific advantages of bacterial concentration might include facilitating the detection of multiple bacterial strains; removal of matrix-associated assay inhibitors; and provision of adequate sample size reduction to allow for the use of representative food sample sizes and/or small media volumes. Furthermore, bacterial concentration could aid in improving sampling techniques needed to detect low levels of pathogens or sporadic contamination, which may perhaps reduce or even eliminate the need for cultural enrichment prior to detection. Although bacterial concentration methods such as centrifugation, filtration, and immunomagnetic separation have been reported for food systems, none of these is ideal and in many cases a technique optimized for one food system or microorganism is not readily adaptable to others. Indeed, the separation and subsequent concentration of bacterial cells from a food sample during sample preparation continues to be a stumbling block in the advancement of molecular methods for the detection of foodborne pathogens. The purpose of this review is to provide a detailed understanding of the science, possibilities, and limitations of separating and concentrating bacterial cells from the food matrix in an effort to further improve our ability to harness molecular methods for the rapid detection of foodborne pathogens.
Bacteria↗
Multiplex nucleic acid sequence-based amplification for simultaneous detection of several enteric viruses in model ready-to-eat foods.
Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10(-1) reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 10(0) to 10(2) reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods.
Animals↗
Antimicrobial resistance of Enterococcus species isolated from produce.
The purpose of this study was to characterize the antibiotic resistance profiles of Enterococcus species isolated from fresh produce harvested in the southwestern United States. Among the 185 Enterococcus isolates obtained, 97 (52%) were Enterococcus faecium, 38 (21%) were Enterococcus faecalis, and 50 (27%) were other Enterococcus species. Of human clinical importance, E. faecium strains had a much higher prevalence of resistance to ciprofloxacin, tetracycline, and nitrofurantoin than E. faecalis. E. faecalis strains had a low prevalence of resistance to antibiotics used to treat E. faecalis infections of both clinical and of agricultural relevance, excluding its intrinsic resistance patterns. Thirty-four percent of the isolates had multiple-drug-resistance patterns, excluding intrinsic resistance. Data on the prevalence and types of antibiotic resistance in Enterococcus species isolated from fresh produce may be used to describe baseline antibiotic susceptibility profiles associated with Enterococcus spp. isolated from the environment. The data collected may also help elucidate the role of foods in the transmission of antibiotic-resistant strains to human populations.
Anti-Bacterial Agents↗
Epidemic clone I-specific genetic markers in strains of Listeria monocytogenes serotype 4b from foods.
Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis.
Base Composition↗
Diagnosis of norwalk virus infection by indirect enzyme immunoassay detection of salivary antibodies to recombinant norwalk virus antigen.
Simple diagnostic tests are needed for the detection of norovirus (NoV) outbreaks. Salivary antibody assays provide an attractive alternative to collecting and testing serum or stool samples. Antibodies to Norwalk virus (NV) in oral fluid samples were compared with NV antibodies in serum collected from 38 volunteers challenged with NV inoculum. Pre- and postchallenge (day 4, 8, 14, and 21) saliva and serum samples were examined by enzyme immunoassay (EIA) using recombinant NV antigen. Of 18 infected subjects (those who shed NV in stool or who demonstrated immunoglobulin G [IgG] seroconversion), 15 (83%) had > or =4-fold increases in NV-specific salivary IgA and 15 (83%) had > or =4-fold increases in NV-specific salivary IgG when prechallenge and postchallenge saliva samples were compared. When the results of the IgA and IgG assays were combined, all 18 infected subjects showed > or =4-fold increases in NV-specific salivary IgG or IgA postchallenge titers compared to their prechallenge titers. One of 19 uninfected subjects had a > or =4-fold increase in NV-specific salivary IgG. The sensitivity of the combined assay results was 100%, and the specificity was 95%. NV-specific salivary IgA titers peaked around 14 days postchallenge. NV-specific salivary IgG and serum IgG titers continued to rise through 21 days postchallenge. The application of this EIA to an elementary school outbreak indicated that 67% of the subjects with confirmed infections had >4-fold rises in anti-NoV IgA when an antigen in the same genetic cluster as the outbreak virus was used. This is the first documented mucosal antibody response to NoV in children. This EIA provides a useful approach for diagnosing NoV outbreaks.
Adult↗
Transcriptional enhancement of RT-PCR for rapid and sensitive detection of Noroviruses.
Previously reported nucleic acid sequence-based amplification (NASBA) primers specific for the GII Noroviruses were adapted for reverse transcriptase-polymerase chain reaction (RT-PCR), and detection sensitivity was then enhanced by a subsequent in vitro transcription of the RT-PCR amplicons. The NASBA-derived primers performed comparably to other broadly reactive GII Norovirus primers with respect to detection limits (i.e. 1 RT-PCR amplifiable unit (RT-PCRU) per reaction). Detection limits improved by approximately 1 log(10) to 0.3 RT-PCRU per reaction when transcriptional enhancement and electrochemiluminescence (ECL) hybridization followed RT-PCR. The method shows promise for improved detection sensitivity in instances where very low levels of virus contamination might be anticipated.
Environmental Microbiology↗