In-House Validation of a Rinse–Membrane Filtration Method for Processing Fresh Produce Samples for Downstream Cultural Detection of Salmonella, Escherichia coli O157:H7, and Listeria

2020 ◽  
Vol 83 (9) ◽  
pp. 1592-1597
Author(s):  
LAURA E. TIJERINA-RODRÍGUEZ ◽  
LUISA SOLÍS-SOTO ◽  
NORMA HEREDIA ◽  
JUAN S. LEÓN ◽  
LEE-ANN JAYKUS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Filtration ◽  
Pathogen Detection ◽  
Fresh Produce ◽  
Bacterial Pathogen ◽  
Relative Sensitivity ◽  
Good Alternative ◽  
Detection Methods ◽  
Filtration Method ◽  
Relative Specificity

ABSTRACT More efficient sampling and detection methods of pathogens on fresh produce are needed. The purpose of this study was to compare a novel rinse–membrane filtration method (RMFM) to a more traditional sponge rubbing or stomaching method in processing jalapeño peppers and cantaloupe samples for detection of Escherichia coli, Salmonella enterica, and Listeria monocytogenes. For jalapeño peppers inoculated with 106, 104, and 102 CFU of each pathogen and cantaloupes inoculated at 106 and 104 CFU, all pathogens were detected in all (100%) samples by RMFM at a 10-mL filtration volume, as well as by the stomacher and sponge rubbing methods. However, for cantaloupe inoculated at 102 CFU, detection differed by pathogen: S. enterica (20% RMFM, 60% stomacher, and 20% sponge), L. monocytogenes (40% RMFM, 60% stomacher, and 20% sponge), and E. coli O157:H7 (100% RMFM, 75% stomacher, and 75% sponge). When RMFM was compared with the other methods, in accordance with guidelines in the International Organization for Standardization 16140:2003 protocol, it produced values >95% in relative accuracy, relative specificity, and relative sensitivity. Overall, the RMFM performed similar to or better than the homogenization and sponge surface rubbing methods and is a good alternative for processing large numbers of produce samples for bacterial pathogen detection.

Detection of Campylobacter jejuni from fresh produce: comparison of culture- and PCR-based techniques, and metagenomic approach for analyses of the microbiome before and after enrichment

2021 ◽  
Author(s):  
Jung-Whan Chon ◽  
Ji Young Jung ◽  
Youngbeom Ahn ◽  
Dongryeoul Bae ◽  
Saeed Khan ◽  
...  
Keyword(s):  
Real Time ◽  
Campylobacter Jejuni ◽  
Membrane Filtration ◽  
Detection Efficiency ◽  
Fresh Produce ◽  
Culture Method ◽  
Detection Methods ◽  
Filtration Method ◽  
Significant Difference ◽  
Alfalfa Sprouts

In this study, we compared the efficiency of culture-based methods with or without membrane filtration, real-time PCR and digital droplet PCR (ddPCR) for the detection of Campylobacter in fresh produce. Alfalfa sprouts, clover sprouts, coleslaw, and lettuce salad spiked with Campylobacter jejuni ( C. jejuni ) were enriched in Bolton broth for 48 h and enrichment cultures were either directly inoculated onto modified charcoal-cefoperazone-deoxycholate agar or applied on membrane filters placed on the surface of plating media. In parallel, 2 mL Bolton broth cultures were taken to extract DNA for real-time and ddPCR assays and bacterial community analysis. There was no significant difference ( p > 0.05) in the detection efficiency of positive Campylobacter isolates from coleslaw and lettuce salad using four detection methods. However, for sprout samples, the detection efficiency of the culture method was significantly ( p < 0.05) lower than those of the two PCR assays and the filtration method. The analysis also revealed the presence of Pseudomonas and Acinetobacter as the most prevalent competing microbiota in enriched culture and only Acinetobacter on agar plates in the selective culture step.

An Improved Membrane Filtration Method for Enumeration of Faecal Coliforms and E. Coli by a Fluorogenic β-Glucuronidase Assay

1993 ◽  
Vol 27 (3-4) ◽  
pp. 267-270 ◽  
Author(s):  
M. T. Augoustinos ◽  
N. A. Grabow ◽  
B. Genthe ◽  
R. Kfir
Keyword(s):  
Escherichia Coli ◽  
Water Samples ◽  
Membrane Filtration ◽  
Faecal Coliforms ◽  
Environmental Waters ◽  
Filtration Method ◽  
E Coli ◽  
Wide Range ◽  
Pure Cultures ◽  
Glucuronidase Assay

A fluorogenic β-glucuronidase assay comprising membrane filtration followed by selective enumeration on m-FC agar at 44.5°C and further confirmation using tlie 4-metliylumbelliferyl-β-D-glucuronide (MUG) containing medium was evaluated for the detection of Escherichia coli in water. A total of 200 typical blue and non-typical blue colonies were isolated from sea and fresh water samples using initial selective enumeration on m-FC agar. Pure cultures of the selected colonies were further tested using the MUG assay and identified using the API 20E method. Of the colonies tested which were shown to be positive using the MUG assay 99.4% were Escherichia coli. The results of this study indicate the combination of the m-FC method followed by the MUG assay to be highly efficient for the selection and confirmation of E. coli from a wide range of environmental waters.

scholarly journals Application of quantitative real-time PCR compared to filtration methods for the enumeration of Escherichia coli in surface waters within Vietnam

2016 ◽  
Vol 15 (1) ◽  
pp. 155-162 ◽  
Author(s):  
Pierangeli G. Vital ◽  
Nguyen Thi Van Ha ◽  
Le Thi Hong Tuyet ◽  
Kenneth W. Widmer
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Surface Waters ◽  
Real Time Pcr ◽  
Membrane Filtration ◽  
Quantitative Real Time Pcr ◽  
Wet Season ◽  
Culture Methods ◽  
Fecal Indicator ◽  
Filtration Method

Surface water samples in Vietnam were collected from the Saigon River, rural and suburban canals, and urban runoff canals in Ho Chi Minh City, Vietnam, and were processed to enumerate Escherichia coli. Quantification was done through membrane filtration and quantitative real-time polymerase chain reaction (PCR). Mean log colony-forming unit (CFU)/100 ml E. coli counts in the dry season for river/suburban canals and urban canals were log 2.8 and 3.7, respectively, using a membrane filtration method, while using Taqman quantitative real-time PCR they were log 2.4 and 2.8 for river/suburban canals and urban canals, respectively. For the wet season, data determined by the membrane filtration method in river/suburban canals and urban canals samples had mean counts of log 3.7 and 4.1, respectively. While mean log CFU/100 ml counts in the wet season using quantitative PCR were log 3 and 2, respectively. Additionally, the urban canal samples were significantly lower than those determined by conventional culture methods for the wet season. These results show that while quantitative real-time PCR can be used to determine levels of fecal indicator bacteria in surface waters, there are some limitations to its application and it may be impacted by sources of runoff based on surveyed samples.

Relative Sensitivity of Escherichia coli O157 Detection from Bovine Feces and Rectoanal Mucosal Swabs

2014 ◽  
Vol 77 (6) ◽  
pp. 972-976 ◽  
Author(s):  
K. J. WILLIAMS ◽  
M. P. WARD ◽  
O. DHUNGYEL ◽  
L. VAN BREDA
Keyword(s):  
Escherichia Coli ◽  
Confidence Interval ◽  
Human Health Risk ◽  
Relative Sensitivity ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Escherichia Coli O157 ◽  
Prevalence Studies ◽  
E Coli ◽  
The Difference

The need to quantify the potential human health risk posed by the bovine reservoir of Escherichia coli O157 has led to a wealth of prevalence studies and improvements in detection methods over the last two decades. Rectoanal mucosal swabs have been used for the detection of E. coli O157 fecal shedding, colonized animals, and those predisposed to super shedding. We conducted a longitudinal study to compare the detection of E. coli O157 from feces and rectoanal mucosal swabs (RAMS) from a cohort of dairy heifers. We collected 820 samples that were tested by immunomagnetic separation of both feces and RAMS. Of these, 132 were detected as positive for E. coli O157 from both samples, 66 were detected as positive from RAMS only, and 117 were detected as positive from feces only. The difference in results between the two sample types was statistically significant (P < 0.001). The relative sensitivities of detection by immunomagnetic separation were 53% (confidence interval, 46.6 to 59.3) from RAMS and 67% (confidence interval, 59.6 to 73.1) from fecal samples. No association between long-term shedding (P = 0.685) or super shedding (P = 0.526) and detection by RAMS only was observed.

scholarly journals Methods for enumeratingEscherichia coliin subtropical waters

1991 ◽  
Vol 106 (2) ◽  
pp. 345-354 ◽  
Author(s):  
W. H. S. Cheung ◽  
D. K. K. Ha ◽  
K. Y. Yeung ◽  
R. P. S. Hung
Keyword(s):  
Membrane Filtration ◽  
Good Alternative ◽  
Environmental Waters ◽  
Filtration Method ◽  
E Coli ◽  
Bacterial Indicator ◽  
Urease Test ◽  
The Uk ◽  
Mls Method

SUMMARYThe standard membrane filtration method of the UK has been modified in order to improve its specificity for enumeratingEscherichia coliin the subtropical waters of Hong Kong. This involves incorporating into the membrane lauryl sulphate (mLS) method either anin situurease test (the mLS-UA method), or anin situβ-glucuronidase test (the mLS-GUD method). The false-positive errors of the mLS-UA and mLS-GUD methods are low, ranging from 3–5%. A comparison between the membrane filtration (mLS-UA) method and the multiple tube technique in testingE. coliin subtropical beach-waters has demonstrated that the former can give much more precise counts, and is the method of choice for such a purpose. The mLS-GUD method, for which automated counting ofE. colicolonies is possible, is a good alternative to mLS-UA in routine enumeration of this bacterial indicator in environmental waters.

Heparin-Induced Thrombocytopenia: Comparison Between the Standard Gel-Particle Assay (PaGIA) and the Functional Flow Cytometric Assay.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5060-5060
Author(s):  
Aaron Tomer
Keyword(s):  
Conflicts Of Interest ◽  
Multiorgan Failure ◽  
Relative Sensitivity ◽  
Serotonin Release ◽  
Detection Methods ◽  
Antibody Detection ◽  
Flow Cytometric Assay ◽  
Heparin Induced Thrombocytopenia ◽  
Flow Cytometric ◽  
Relative Specificity

Abstract 5060 Background Heparin-induced thrombocytopenia and thrombosis (HIT) is an immune-mediated complication that may develop in patients sensitized to heparin. Approximately 5% of patients treated with full dose heparin develop clinical HIT, with about half develop thrombosis that may be associated with severe morbidity and death. However, antibodies may be detected in up to 30% of patients. Thus, current antibody-detection methods carry certain limitations, which pose a serious clinical dilemma in the diagnosis and treatment of HIT. Objectives To compare the gel-particle immuno-assay (PaGIA) for the detection of antibodies against heparin-PF4 complex, with the functional flow cytometric assay (FCA) which determines the capacity of the patient's serum to activate platelets in the presence of heparin, similar in concept to the gold-standard, the radioactive Serotonin-release assay. Methods Sequential samples from patients clinically suspected for HIT were tested by both PaGIA and the FCA (Tomer 1997, 1999). Results 118 samples were tested. Positive: 9 (7.6%) patients tested positive by PaGIA, compared to 19 (16.1%) by the FCA. 7,out of the 9 (77.8%) PaGIA -positive samples were also positive by the FCA (relative sensitivity). Negative: 97 out of 109 gel-negative samples (89.0%) were also negative by the FCA (relative specificity). Thrombosis occurred in 3 of the 9 PaGIA-positive (33%) patients, and in 7 of the 19 FCA-positive (36.8%) patients. Of the 12 PaGIA -negative but FCA-positive patients, 4 (33%) had thrombosis. Death rate was also higher among FCA-positive (n=3) compared to PaGIA-positive (n=1) patients. Of the two PaGIA -positive but FCA-negative patients, one had APLA syndrome (APS) with chronic thrombocytopenia, and one had sepsis with cardiogenic shock and multiorgan failure. ROC-Plot: Overall, the FCA showed significantly higher correlation with the clinical presentation of HIT (4Ts score), compared to the PaGIA (AUC 0.86 vs. 0.62, p<0.001) Conclusion The functional FCA demonstrates superior sensitivity and specificity compared to the antibody- detection PaGIA gel-particle assay. The feasible functional FCA (results in 1.5 hr) might be useful for initial diagnosis of HIT, and particularly for confirmation of HIT in patients with confounding presentation, including negative antibody-detection assay. Disclosures No relevant conflicts of interest to declare.

scholarly journals Prevalence and Genomic Characterization of Escherichia coli O157:H7 in Cow-Calf Herds throughout California

2017 ◽  
Vol 83 (16) ◽  
Author(s):  
Jay N. Worley ◽  
Kristopher A. Flores ◽  
Xun Yang ◽  
Jennifer A. Chase ◽  
Guojie Cao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Fresh Produce ◽  
Bacterial Pathogen ◽  
Virulence Gene ◽  
The United States ◽  
Pathogenic Potential ◽  
Content Type ◽  
E Coli ◽  
Cow Calf

ABSTRACT Escherichia coli serotype O157:H7 is a zoonotic food- and waterborne bacterial pathogen that causes a high hospitalization rate and can cause life-threatening complications. Increasingly, E. coli O157:H7 infections appear to originate from fresh produce. Ruminants, such as cattle, are a prominent reservoir of E. coli O157:H7 in the United States. California is one of the most agriculturally productive regions in the world for fresh produce, beef, and milk. The close proximity of fresh produce and cattle presents food safety challenges on a uniquely large scale. We performed a survey of E. coli O157:H7 on 20 farms in California to observe the regional diversity and prevalence of E. coli O157:H7. Isolates were obtained from enrichment cultures of cow feces. Some farms were sampled on two dates. Genomes from isolates were sequenced to determine their relatedness and pathogenic potential. E. coli O157:H7 was isolated from approximately half of the farms. The point prevalence of E. coli O157:H7 on farms was highly variable, ranging from zero to nearly 90%. Within farms, generally one or a few lineages were found, even when the rate of isolation was high. On farms with high isolation rates, a single clonal lineage accounted for most of the isolates. Farms that were visited months after the first visit might have had the same lineages of E. coli O157:H7. Strains of E. coli O157:H7 may be persistent for months on farms. IMPORTANCE This survey of 20 cow-calf operations from different regions of California provides an in depth look at resident Escherichia coli O157:H7 populations at the molecular level. E. coli O157:H7 is found to have a highly variable prevalence, and with whole-genome sequencing, high prevalences in herds were found to be due to a single lineage shed from multiple cows. Few repeat lineages were found between farms in this area; therefore, we predict that E. coli O157:H7 has significant diversity in this area beyond what is detected in this survey. All isolates from this study were found to have pathogenic potential based on the presence of key virulence gene sequences. This represents a novel insight into pathogen diversity within a single subtype and will inform future attempts to survey regional pathogen populations.

Antimicrobial Resistance and Genetic Profiling of Escherichia coli from a Commercial Beef Packing Plant†

2006 ◽  
Vol 69 (7) ◽  
pp. 1508-1513 ◽  
Author(s):  
MUEEN ASLAM ◽  
CARA SERVICE
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Membrane Filtration ◽  
Antimicrobial Agents ◽  
Random Amplified Polymorphic Dna ◽  
Ground Beef ◽  
Resistant Bacteria ◽  
Filtration Method ◽  
E Coli ◽  
Beef Processing

The objective of this study was to investigate the extent of antimicrobial resistance and to genetically characterize resistant Escherichia coli recovered from a commercial beef packing plant. E. coli isolates were recovered by a hydrophobic grid membrane filtration method by direct plating on SD-39 medium. A total of 284 isolates comprising 71, 36, 55, 52, and 70 isolates from animal hides, washed carcasses, conveyers, beef trimmings, and ground beef, respectively, were analyzed. The susceptibility of E. coli isolates to 15 antimicrobial agents was evaluated with an automated broth microdilution system, and the genetic characterization of these isolates was performed by the random amplified polymorphic DNA (RAPD) method. Of the 284 E. coli isolates, 56% were sensitive to all 15 antimicrobial agents. Resistance to tetracycline, ampicillin, and streptomycin was observed in 38, 9, and 6% of the isolates, respectively. Resistance to one or more antimicrobial agents was observed in 51% of the E. coli isolates recovered from the hides but in only 25% of the E. coli from the washed carcasses. Resistance to one or more antimicrobial agents was observed in 49, 50, and 37% of the isolates recovered from conveyers, beef trimmings, and ground beef, respectively. The RAPD pattern data showed that the majority of resistant E. coli isolates were genetically diverse. Only a few RAPD types of resistant strains were shared among various sample sources. The results of this study suggest that antimicrobial-resistant E. coli isolates were prevalent during all stages of commercial beef processing and that considerably higher numbers of resistant E. coli were present on conveyers, beef trimmings, and ground beef than on dressed carcasses. This stresses the need for improving hygienic conditions during all stages of commercial beef processing and meatpacking to avoid the risks of transfer of antimicrobial-resistant bacteria to humans.

Removal of Escherichia coli and Enterococcus faecalis after Hand Washing with Antimicrobial and Nonantimicrobial Soap and Persistence of These Bacteria in Rinsates

2017 ◽  
Vol 80 (10) ◽  
pp. 1670-1675 ◽  
Author(s):  
J. Pérez-Garza ◽  
S. García ◽  
N. Heredia
Keyword(s):  
Escherichia Coli ◽  
Enterococcus Faecalis ◽  
Membrane Filtration ◽  
Chlorhexidine Gluconate ◽  
Hand Washing ◽  
Distilled Water ◽  
Antimicrobial Compounds ◽  
Filtration Method ◽  
E Coli ◽  
Reducing Bacteria

ABSTRACT Foodhandlers are important sources of contamination in the agricultural environment. This study was conducted (i) to evaluate the activity of antimicrobial soaps against Escherichia coli and Enterococcus faecalis using a hand washing model with soiled hands and (ii) to determine the survival and persistence of these bacteria in rinsates. Sterilized agricultural soil from tomato and pepper farms was inoculated with E. coli or E. faecalis at 103 or 106 CFU/g. Decontaminated hands were placed in contact with contaminated soil for 2 min and were then washed with soaps with or without antimicrobial compounds (citric extracts, chloroxylenol, triclosan, or chlorhexidine gluconate). As the control, hands were washed with sterile distilled water. The levels of bacteria remaining on the hands and recovered from the rinsates were determined using a membrane filtration method and selective media. Antimicrobial soaps removed levels of E. coli similar to those removed by distilled water and nonantimicrobial soap on hands contaminated with E. coli at 103 CFU/g. However, when hands were contaminated with E. coli at 106 CFU/g, more E. coli was removed with the chlorhexidine gluconate soap. When hands were contaminated with E. faecalis at 103 CFU/g, bacteria were removed more effectively with soaps containing chloroxylenol or chlorhexidine gluconate. When hands were contaminated with E. faecalis at 106 CFU/g, all of the antimicrobial soaps were more effective for removing the bacteria than were distilled water and nonantimicrobial soap. E. coli grew in all of the hand washing rinsates except that containing triclosan, whereas E. faecalis from the 106 CFU/g treatments grew in rinsates containing chlorhexidine gluconate and in the distilled water rinsates. Washing with antimicrobial soap was more effective for reducing bacteria on soiled hands than was washing with water or nonantimicrobial soap. However, persistence or growth of bacteria in these rinsates poses health risks.

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