Enhanced Detection of Listeria spp. in Farmstead Cheese Processing Environments through Dual Primary Enrichment, PCR, and Molecular Subtyping

2008 ◽  
Vol 71 (11) ◽  
pp. 2239-2248 ◽  
Author(s):  
DENNIS J. D'AMICO ◽  
CATHERINE W. DONNELLY
Keyword(s):  
Listeria Monocytogenes ◽  
Environmental Samples ◽  
Environmental Sampling ◽  
Strain Diversity ◽  
Enrichment Protocol ◽  
Automated Ribotyping ◽  
Epidemiological Significance ◽  
Enrichment Methods ◽  
Over Time ◽  
Higher Sensitivity

The incidence and ecology of Listeria spp. in farmstead cheese processing environments were assessed through environmental sampling conducted in nine different plants over a 10-week period. Environmental samples (n = 705) were examined for the presence of Listeria spp. by using three detection/isolation protocols. The use of dual enrichment methods, which allowed for the recovery of injured Listeria spp. (mUSDA), identified more Listeria species–positive samples with higher sensitivity than the standard USDA method. The addition of PCR to the mUSDA method identified the most Listeria monocytogenes–positive samples, achieving greater sensitivity of detection while substantially reducing time. Overall, 7.5% of samples were positive for Listeria spp., yielding 710 isolates, 253 of which were subtyped by automated ribotyping to examine strain diversity within and between plants over time. The isolation of specific ribotypes did not appear to be affected by the enrichment protocol used. Fifteen (2.1%) samples yielded L. monocytogenes isolates differentiated almost equally into ribotypes of lineages I and II. Of most concern was the persistent and widespread contamination of a plant with L. monocytogenes DUP-1042B, a ribotype previously associated with multiple outbreaks of listeriosis. Our results suggest that the extent of contamination by Listeria spp., notably L. monocytogenes, in farmstead cheese plants is comparatively low, especially for those with on-site farms. The results of this study also identified points of control for use in designing more effective Listeria spp. control and monitoring programs with a focus on ribotypes of epidemiological significance.

Contamination Patterns of Listeria monocytogenes in Cold-Smoked Pork Processing

2010 ◽  
Vol 73 (11) ◽  
pp. 2103-2109 ◽  
Author(s):  
AIVARS BĒRZIŅŠ ◽  
SANNA HELLSTRÖM ◽  
INDULIS SILIŅŠ ◽  
HANNU KORKEALA
Keyword(s):  
Listeria Monocytogenes ◽  
Environmental Samples ◽  
Meat Products ◽  
Pulsed Field Gel Electrophoresis ◽  
Processing Plant ◽  
Environmental Sampling ◽  
Meat Processing ◽  
Pork Products ◽  
Plant Environment ◽  
Pork Samples

Contamination patterns of Listeria monocytogenes were studied in a cold-smoked pork processing plant to identify the sources and possible reasons for the contamination. Environmental sampling combined with pulsed-field gel electrophoresis (PFGE) subtyping and serotyping were applied to investigate the genetic diversity of L. monocytogenes in the plant environment and ready-to-eat (RTE) cold-smoked pork products. A total of 183 samples were collected for contamination analyses, including samples of the product at different stages during manufacture (n = 136) and environmental samples (n = 47) in 2009. L. monocytogenes isolates, previously recovered from 73 RTE cold-smoked pork samples and collected from the same meat processing plant in 2004, were included in this study. The brining machine and personnel working with brining procedures were the most contaminated places with L. monocytogenes. The overall prevalence of L. monocytogenes in raw pork (18%) increased to 60% after the brining injections. The brining machine harbored six different PFGE types belonging to serotypes 1/2a, 1/2c, 4b, and 4d, which were found on the feeding teeth, smooth surfaces, and spaces of the machine, thus potentially facilitating dissemination of L. monocytogenes contamination. Two PFGE types (2 and 8) belonging to serotypes 1/2a and 1/2c were recovered from RTE cold-smoked pork collected in 2004, and from surfaces of the brining machine sampled in 2009, and may indicate the presence of persistent L. monocytogenes strains in the plant. Due to poor hygiene design, removal of the brining machine from the production of cold-smoked meat products should be considered to reduce L. monocytogenes contamination in the finished products.

Application of Automated Ribotyping To Support the Evaluation of Listeria monocytogenes Sources in a Taleggio Cheese Producing Plant

2007 ◽  
Vol 70 (5) ◽  
pp. 1116-1121 ◽  
Author(s):  
A. DE CESARE ◽  
G. MANFREDA ◽  
M. MACRÌ ◽  
C. CANTONI
Keyword(s):  
Listeria Monocytogenes ◽  
Molecular Typing ◽  
Environmental Samples ◽  
Processing Plant ◽  
Significant Incidence ◽  
Serotype 1 ◽  
Automated Ribotyping

In March 2005, Listeria monocytogenes was detected on the rinds of Taleggio cheeses produced in an Italian plant. To identify the pathogen source, 154 rinds of cheeses that had been manually and automatically salinated and 52 environmental swabs collected from salting equipment, ripening cloths, and ripening boxes were tested for L. monocytogenes. Twenty-seven strains isolated from cheese samples and 16 strains isolated from environmental samples were genotyped by EcoRI and PvuII automated ribotyping. The microbiological results revealed a significant incidence of contamination of cheeses that were automatically salinated and contamination on the salting equipment, ripening cloths, and boxes. All cheese and environmental strains had the same EcoRI and PvuII ribotyping profiles, designated 153-204-S5 and 153-210-S-2, respectively. The only exception were three Taleggio strains, isolated from the same lot of product, that had EcoRI and PvuII ribotyping profiles designated 153-289-S6 and 153-214-S-5, respectively. Strains with EcoRI profile 153-204-S5 were classified as DUP-ID 1045 and serotype 1/2a, whereas strains with EcoRI profile 153-289-S6 were classified as DUP-ID 1034 and serotype 1/2b. The microbiological and molecular typing data collected in this study suggest that the source of the L. monocytogenes contamination in the Taleggio plant under study was the automated salting equipment. The isolate DUP-IDs were used to trace the introduction of potentially dangerous strains, such as those characterized as DUP-ID 1034, in the processing plant.

scholarly journals Molecular Studies on the Ecology of Listeria monocytogenes in the Smoked Fish Processing Industry

2001 ◽  
Vol 67 (1) ◽  
pp. 198-205 ◽  
Author(s):  
Dawn M. Norton ◽  
Meghan A. McCamey ◽  
Kenneth L. Gall ◽  
Janet M. Scarlett ◽  
Kathryn J. Boor ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Food Processing ◽  
Environmental Samples ◽  
Raw Materials ◽  
Raw Material ◽  
Fish Processing ◽  
Culture Methods ◽  
Smoked Fish ◽  
Molecular Approaches ◽  
Automated Ribotyping

ABSTRACT We have applied molecular approaches, including PCR-based detection strategies and DNA fingerprinting methods, to study the ecology ofListeria monocytogenes in food processing environments. A total of 531 samples, including raw fish, fish during the cold-smoking process, finished product, and environmental samples, were collected from three smoked fish processing facilities during five visits to each facility. A total of 95 (17.9%) of the samples tested positive forL. monocytogenes using a commercial PCR system (BAX for Screening/Listeria monocytogenes), including 57 (27.7%) environmental samples (n = 206), 8 (7.8%) raw material samples (n = 102), 23 (18.1%) samples from fish in various stages of processing(n = 127), and 7 (7.3%) finished product samples (n= 96). L. monocytogenes was isolated from 85 samples (16.0%) using culture methods. Used in conjunction with a 48-h enrichment in Listeria Enrichment Broth, the PCR system had a sensitivity of 91.8% and a specificity of 96.2%. To track the origin and spread of L. monocytogenes, isolates were fingerprinted by automated ribotyping. Fifteen different ribotypes were identified among 85 isolates tested. Ribotyping data established possible contamination patterns, implicating raw materials and the processing environment as potential sources of finished product contamination. Analysis of the distribution of ribotypes revealed that each processing facility had a unique contamination pattern and that specific ribotypes persisted in the environments of two facilities over time (P ≤ 0.0006). We conclude that application of molecular approaches can provide critical information on the ecology of different L. monocytogenes strains in food processing environments. This information can be used to develop practical recommendations for improved control of this important food-borne pathogen in the food industry.

Low Potential Virulence Associated with Mutations in the inlA and prfA Genes in Listeria monocytogenes Isolated from Raw Retail Poultry Meat

2013 ◽  
Vol 76 (1) ◽  
pp. 129-132 ◽  
Author(s):  
VICTORIA LÓPEZ ◽  
JAIME NAVAS ◽  
JOAQUÍN V. MARTÍNEZ-SUÁREZ
Keyword(s):  
Listeria Monocytogenes ◽  
Gel Electrophoresis ◽  
Virulence Genes ◽  
Pulsed Field Gel Electrophoresis ◽  
Poultry Meat ◽  
Pathogenic Potential ◽  
Molecular Serotyping ◽  
Potential Source ◽  
Raw Foods ◽  
Over Time

Packaged raw foods can represent a potential source of Listeria monocytogenes contamination when opened at home, and listeriosis is associated with the consumption of undercooked raw foods. The aim of this study was to characterize a group of L. monocytogenes strains isolated from 56 packages of raw chicken meat from a single brand in order to determine the diversity of the strains that dominate in a particular food over time, as well as their pathogenic potential. Forty (71%) samples were found to be positive for L. monocytogenes, and three isolates per sample were subjected to PCR molecular serotyping. Subtyping of 45 isolates from different manufacturing dates (n = 40) or different molecular serotype within the same sample (n = 5) identified 11 different L. monocytogenes subtypes as defined by pulsed-field gel electrophoresis and sequencing of virulence genes actA and inlA. Two of the subtypes accounted for 51% of the isolates. About 40% of isolates (three subtypes) were found to potentially present attenuated virulence because of the presence of mutations in the prfA and inlA genes.

Some Chemical and Bacteriological Characteristics of Regional Cheeses from Asturias, Spain

1996 ◽  
Vol 59 (5) ◽  
pp. 509-515 ◽  
Author(s):  
ABELARDO MARGOLLES ◽  
ANA RODRIGUEZ ◽  
CLARA G. de los REYES-GAVILAN
Keyword(s):  
Listeria Monocytogenes ◽  
Listeria Innocua ◽  
Chemical Characteristics ◽  
Salmonella Spp ◽  
Cheese Making ◽  
Ph Values ◽  
Plate Counts ◽  
Coagulase Positive Staphylococci ◽  
Enrichment Methods

One hundred and one samples of six representative short-ripened cheeses (five homemade and one industrially manufactured) were collected over 1 year in several supermarkets in Asturias and analyzed for mesophilic plate counts, coliforms, enterobacteria, coagulase-positive staphylococci, the presence of species of Salmonella and Listeria, pH, moisture, NaCl, and aw. Chemical characteristics varied, largely depending on the type of cheese. The percentages of moisture and NaCl ranged from 36.11 to 48.91 and from 1.16 to 2.08 respectively. The aw values were between 0.95 and 0.99. Acidification was quite efficient, all cheeses having mean pH values between 4.56 and 5.39. None of the samples yielded Salmonella spp. Coagulase-positive staphylococci were detected in two cheeses, in one reaching levels up to 106 CFU/g. Listeria spp. contaminated 11.8% of the cheeses, with Listeria monocytogenes isolated from 8.91 % and Listeria innocua from 4.95% of the samples. The distribution of Listeria spp. varied largely depending on the type of cheese: 41% of the samples contaminated with L. monocytogeneswere obtained from one type of cheese which had the lowest pH and NaCl values and the highest aw and moisture levels of the cheeses analyzed. However, L. monocytogenes was absent from another type of cheese, which showed intermediate chemical characteristics. High levels of coliforms and enterobacteria (4 to 5 log CFU/ml) were detected in the five homemade cheeses and were statistically associated with the presence of Listeria spp. and L. monocytogenes. Cold enrichment was unsuccessful for the recovery of Listeria spp. from the cheeses analyzed, while a combination of different enrichment methods resulted in the best procedure for detecting all positive samples. This study shows that L. monocytogenes and coagulase-positive staphylococci are present in short-ripened cheeses consumed in Asturias. Adequate measures to prevent contamination during cheese making will probably result in safer products.

A Canadian Comparative Study of Modified Versions of the “FDA” and “USDA” Methods for the Detection of Listeria monocytogenes

1991 ◽  
Vol 54 (9) ◽  
pp. 669-676 ◽  
Author(s):  
DONALD W. WARBURTON ◽  
JEFFREY M. FARBER ◽  
ANDREW ARMSTRONG ◽  
RICARDO CALDEIRA ◽  
NARAYAN P. TIWARI ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Comparative Study ◽  
Lithium Chloride ◽  
Environmental Samples ◽  
Screening Tool ◽  
Significant Difference ◽  
Better Than

Eleven laboratories across Canada took part in a comparative study of modified versions of the “FDA” and “USDA” methods for the detection of Listeria monocytogenes in foods and environmental samples. Both were modified by the inclusion of additional plating media and the use of modified Fraser broth in the modified “FDA” method. Approximately 92% of the positive samples were detected after 24 h of enrichment. Testing of routine samples by the participating laboratories showed no significant difference (p<0.05) in ability to isolate L. monocytogenes by either the modified “FDA” or “USDA” methods. However, the modified “FDA” method isolated significantly more positives (16.8%) from the spiked foods/controls than the modified “USDA” method (p<0.05). For all samples tested by both methods in the same laboratory, again the modified “FDA” method significantly out performed the “USDA” version by approximately 6% (p<0.05). However, the spiked foods/controls tested by both methods in the same laboratory showed no difference (p<0.05) in their ability to isolate L. monocytogenes. Overall, the modified “FDA” and “USDA” methods were comparable (within 1.0%) in their ability to isolate this microorganism. The “USDA” preenrichment broth maintained its initial pHbetter. Modified Fraser broth, in principle, proved to be useful as a screening tool but is not very selective. Oxford agar proved to be marginally better than lithium chloride-phenylethanol-moxalactam medium and significantly (p<0.05) better than modified Oxford agar in isolating L. monocytogenes.

Listeria monocytogenes Contamination Patterns for the Smoked Fish Processing Environment and for Raw Fish

2003 ◽  
Vol 66 (1) ◽  
pp. 52-60 ◽  
Author(s):  
ADAM D. HOFFMAN ◽  
KENNETH L. GALL ◽  
DAWN M. NORTON ◽  
MARTIN WIEDMANN
Keyword(s):  
Listeria Monocytogenes ◽  
Environmental Contamination ◽  
West Coast ◽  
Environmental Samples ◽  
Raw Materials ◽  
Processing Plant ◽  
Fish Processing ◽  
Smoked Fish ◽  
Fish Samples ◽  
Raw Fish

Reliable data on the sources of Listeria monocytogenes contamination in cold-smoked fish processing are crucial in designing effective intervention strategies. Environmental samples (n = 512) and raw fish samples (n = 315) from two smoked fish processing facilities were screened for L. monocytogenes, and all isolates were subtyped by automated ribotyping to examine the relationship between L. monocytogenes contamination from raw materials and that from environmental sites. Samples were collected over two 8-week periods in early spring and summer. The five types of raw fish tested included lake whitefish, sablefish, farm-raised Norwegian salmon, farm-raised Chilean salmon, and feral (wild-caught) salmon from the U.S. West Coast. One hundred fifteen environmental samples and 46 raw fish samples tested positive for L. monocytogenes. Prevalence values for environmental samples varied significantly (P < 0.0001) between the two plants; plant A had a prevalence value of 43.8% (112 of 256 samples), and plant B had a value of 1.2% (3 of 256 samples). For plant A, 62.5% of drain samples tested positive for L. monocytogenes, compared with 32.3% of samples collected from other environmental sites and 3.1% of samples collected from food contact surfaces. Ribotyping identified 11 subtypes present in the plant environments. Multiple subtypes, including four subtypes not found on any raw fish, were found to persist in plant A throughout the study. Contamination prevalence values for raw fish varied from 3.6% (sablefish) to 29.5% (U.S. West Coast salmon), with an average overall prevalence of 14.6%. Sixteen separate L. monocytogenes subtypes were present on raw fish, including nine that were not found in the plant environment. Our results indicate a disparity between the subtypes found on raw fish and those found in the processing environment. We thus conclude that environmental contamination is largely separate from that of incoming raw materials and includes strains persisting, possibly for years, within the plant. Operational and sanitation procedures appear to have a significant impact on environmental contamination, with both plants having similar prevalence values for raw materials but disparate contamination prevalence values for the environmental sites. We also conclude that regular L. monocytogenes testing of drains, combined with molecular subtyping of the isolates obtained, allows for efficient monitoring of persistent L. monocytogenes contamination in a processing plant.

scholarly journals Comparison of three neutralizing broths for environmental sampling of low levels of Listeria monocytogenes desiccated on stainless steel surfaces and exposed to quaternary ammonium compounds

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Fengmin Li ◽  
Zhihan Xian ◽  
Hee Jin Kwon ◽  
Jiyoon Yoo ◽  
Laurel Burall ◽  
...  
Keyword(s):  
Stainless Steel ◽  
Listeria Monocytogenes ◽  
Quaternary Ammonium ◽  
Storage Conditions ◽  
Ammonium Compound ◽  
Environmental Sampling ◽  
High Concentration ◽  
Environmental Test ◽  
Steel Surfaces ◽  
Low Levels

Abstract Background An effective environmental sampling method involves the use of a transport/neutralizing broth with the ability to neutralize sanitizer residues that are collected during sampling and to maintain viability of stressed Listeria monocytogenes (Lm) cells. Results We applied Lm onto stainless steel surfaces and then subjected Lm to desiccation stress for 16–18 h at room temperature (RT, 21–24 °C). This was followed by the subsequent application of Whisper™ V, a quaternary ammonium compound (QAC)-based sanitizer, diluted to 400 ppm and 8000 ppm of active quat, for 6 h. We then sampled Lm with sponges pre-moistened in three transport broths, Dey/Engley (D/E) broth, Letheen broth and HiCap™ broth, to generate environmental samples that contained sanitizer residues and low levels of stressed Lm, which were subsequently analyzed by an enrichment-based method. This scheme conformed with validation guidelines of AOAC International by using 20 environmental test portions per broth that contained low levels of Lm such that not all test portions were positive (i.e., fractional positive). We showed that D/E broth, Letheen broth and HiCap™ broth performed similarly when no quat or 400 ppm of quat was applied to the Lm contaminating stainless steel surfaces. However, when 8000 ppm of quat was applied, Letheen broth did not effectively neutralize the QAC in the samples. These comparisons were performed on samples stored under three conditions after collection to replicate scenarios of sample transport, RT for 2 h, 4 °C for 24 h and 4 °C for 72 h. Comparisons under the three different scenarios generally reached the same conclusions. In addition, we further demonstrated that storing Letheen and HiCap™ broths at RT for two months before sampling did not reduce their capacity to neutralize sanitizers. Conclusions We developed a scheme to evaluate the ability of transport broths to neutralize QAC sanitizers. The three transport broths performed similarly with a commonly used concentration of quat, but Letheen broth could not effectively neutralize a very high concentration of QAC. The performance of transport broths was not significantly affected under the assessed pre-sampling and post-sampling storage conditions.

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