Delivery of Selective Agents via Time-Delayed Release Tablets Improves Recovery of Listeria monocytogenes Injured by Acid and Nitrite

2014 ◽  
Vol 77 (5) ◽  
pp. 772-780 ◽  
Author(s):  
ESMOND NYARKO ◽  
DENNIS D'AMICO ◽  
PATRICK MACH ◽  
WENSHENG XIA ◽  
CATHERINE DONNELLY
Keyword(s):  
Listeria Monocytogenes ◽  
Cell Populations ◽  
Enrichment Broth ◽  
Selective Enrichment ◽  
Delayed Release ◽  
Selective Agents ◽  
Meat Samples ◽  
Food Sample Analysis ◽  
University Of Vermont ◽  
The U.S

Listeria selective enrichment media are designed to enhance the isolation of the organism and increase the chances of detection. Drawbacks include the requirements for prolonged sample incubation (48 to 72 h) and manual addition of selective agents, which may be a source of contamination. Modified Listeria recovery broth (mLRB) is a proprietary enrichment medium formulated to facilitate the recovery of injured cells; its selective agents are incorporated into a format that allows delayed release until 6 h of incubation. We evaluated the change in cell populations over time for acid- and nitrite-injured Listeria monocytogenes in mLRB with the selective agents added manually at 0 h (mLRBS0) and 6 h (mLRBS6). Recovery of injured cells in mLRB plus time-delayed tablets (mLRBTD) was also compared with that in enrichment media recommended by the U.S. Department of Agriculture (University of Vermont broth), the U.S. Food and Drug Administration (buffered Listeria enrichment broth), and the International Organization for Standardization (demi-Fraser broth). Nitrite- or acid-injured Listeria at approximately 10 CFU/ml were inoculated into each broth medium, and Listeria populations were enumerated at various times from 12 to 48 h of incubation at 37°C. Analysis of variance revealed that acid-injured Listeria populations in mLRBS6 at 24 h were significantly higher (P < 0.05) than those in mLRBS0; however, the differences in populations on these two media were not significant for nitrite-injured cells. Cell populations of four strains of Listeria inoculated into mLRBTD were significantly higher at 24 h than when those strains were enriched in buffered Listeria enrichment broth, demi-Fraser broth, and University of Vermont broth. Comparison between artificially contaminated milk and meat samples with a four-strain cocktail of Listeria resulted in cell populations that were significantly higher (P < 0.05) at 24 h on mLRBTD for contaminated meat than on mLRB for contaminated milk. Delivery of selective agents via time-delayed release tablets into mLRB maximizes recovery of acid- and nitrite-injured Listeria and saves analyst time during food sample analysis.

Quantitative Evaluation of Three Selective Enrichment Broths and Agars Used in Recovering Listeria Microorganisms

1990 ◽  
Vol 53 (2) ◽  
pp. 105-110 ◽  
Author(s):  
JOSE FERNANDEZ-GARAYZABAL ◽  
CONSTANTIN GEMGEORGIS
Keyword(s):  
Brain Heart Infusion ◽  
Superior Performance ◽  
Enrichment Broth ◽  
Selective Enrichment ◽  
Naturally Occurring ◽  
Listeria Ivanovii ◽  
Soft Cheese ◽  
Meat Samples ◽  
Listeria Seeligeri ◽  
University Of Vermont

Three selective enrichment broths (FDA, University of Vermont and Dominguez Rodriguez) and lithium chloride-phenylethanol-moxolactam (LPM) agar, modified McBride (MMA) agar, Listeria selective agar (LSAM of Dominguez-Rodriguez), and Brain Heart Infusion (BHI) agar (as reference) were evaluated for their suitability to support the growth of six different species/strains of Listeria (Listeria monocytogenes Scott A, L. monocytogenes V7, L. monocytogenes VPH-1, Listeria innocua, Listeria seeligeri, and Listeria ivanovii). All Listeria strains grew faster and yielded a higher number of cells in FDA enrichment broth. Based on MPN studies, 1 to 2.1 cells of L. monocytogenes and L. innocua were needed for visible colony formation, on all three selective and BHI agars after 48 h incubation of 37°C. The LPM agar was more inhibitory for L. seeligeri and L. ivanovii requiring 9.6 and 917 cells, respectively, as compared to 1 to 2.7 cells for the other agars. The effectiveness of a particular combination among the selective enrichment broths and agars for recovering L. monocytogenes Scott A from inoculated cheese and meat samples was quantitated. Any enrichment broth combined with plating on LPM or LSAM agar gave 100% Listeria recovery as compared to 50 to 67% for plating on MMA agar. Both LPM and LSAM agars have also shown a superior performance to MMA agar in the recovery of naturally occurring Listeria from soft cheese and raw meat. The use of a secondary broth enrichment step improved the recovery of Listeria spp. from meat samples.

Comparison of Growth Kinetics for Healthy and Heat-Injured Listeria monocytogenes in Eight Enrichment Broths

2002 ◽  
Vol 65 (8) ◽  
pp. 1333-1337 ◽  
Author(s):  
TODD M. SILK ◽  
TATIANA M. T. ROTH ◽  
C. W. DONNELLY
Keyword(s):  
Listeria Monocytogenes ◽  
Growth Curves ◽  
Lag Phase ◽  
Phase Duration ◽  
Enrichment Broth ◽  
Selective Enrichment ◽  
Gompertz Equation ◽  
Selective Agents ◽  
Time Required ◽  
Maximum Population Density

Detection of Listeria in food products is often limited by performance of enrichment media used to support growth of Listeria to detectable levels. In this study, growth curves were generated using healthy and heat-injured Listeria monocytogenes strain F5069 in three nonselective and five selective enrichment broths. Nonselective enrichment media included the current Food and Drug Administration Bacteriological Analytical Manual Listeria enrichment broth base (BAM), Listeria repair broth (LRB), and Trypticase soy broth. Selective enrichment media included BAM with selective agents and LRB with selective agents, BCM L. monocytogenes preenrichment broth, Fraser broth, and UVM-modified Listeria enrichment broth. The Gompertz equation was used to model the growth of L. monocytogenes. Gompertz parameters were used to calculate exponential growth rate, lag-phase duration (LPD), generation time, maximum population density (MPD), and time required for repair of injured cells. Statistical differences (P < 0.05) in broth performance were noted for LPD and MPD when healthy and injured cells were inoculated into the broths. With the exception of Fraser broth, there were no significant differences in the time required for the repair of injured cells. Results indicate that the distinction between selective and nonselective broths in their ability to grow healthy Listeria and to repair sublethally injured cells is not solely an elementary issue of presence or absence of selective agents.

Molecular Characterization of Listeria monocytogenes in white meat samples from Punjab, India

2017 ◽  
Author(s):  
Simranpreet Kaur ◽  
Randhir Singh ◽  
Mandeep Kaur Sran ◽  
J.P. S. Gill
Keyword(s):  
Public Health ◽  
Listeria Monocytogenes ◽  
White Meat ◽  
Selective Enrichment ◽  
Retail Outlets ◽  
Meat Samples ◽  
Pcr Targeting ◽  
University Of Vermont

A study was undertaken to assess the prevalence of Listeria monocytogenes in white meats in the Punjab, India. A total of 335 samples including 115 samples of chicken, 75 samples of pork and 145 samples of fish were collected from retail outlets. Isolation of the pathogen was done by selective enrichment in University of Vermont Medium I and II and plating onto PALCAM agar. The recovered Listeria isolates were subjected to in-vitro pathogenicity assays and multiplex PCR targeting virulence-associated genes (prfA, plcA, actA, hlyA and iap) of L. monocytogenes. A total of thirty one (31) L. monocytogenes strains were isolated from meat samples with the prevalence of L. monocytogenes as 9.2%. Maximum prevalence was seen in fish (19.3%) followed by chicken (1.7%) and pork (1.3%). All isolates recovered from the study exhibited pathogenicity in in-vitro pathogenicity assays as well as possessed all the virulence related genes. Thus, the presence of pathogenic strains of L. monocytogenes in white meats with a prevalence of 9.2% appeared to be a cause for concern with profound public health implications.

scholarly journals Contribution of Selective Conditions to Microbial Competition in Four Listeria Selective Enrichment Formulations

2016 ◽  
Vol 79 (11) ◽  
pp. 1904-1910 ◽  
Author(s):  
ASHLEY L. KEYS ◽  
ANTHONY D. HITCHINS ◽  
R. DERIKE SMILEY
Keyword(s):  
Incubation Temperature ◽  
Competitive Growth ◽  
Microbial Competition ◽  
Enrichment Broth ◽  
Selective Enrichment ◽  
Selective Agents ◽  
Individual Contributions ◽  
The Individual ◽  
University Of Vermont ◽  
Competition Assays

ABSTRACT Microbial competition during selective enrichment negatively affects Listeria monocytogenes populations and may hinder the subsequent detection or recovery of this organism. Competition assays among 10 selected strains of Listeria and Citrobacter braakii were performed in buffered Listeria enrichment broth, 3-(N-morpholino)propanesulfonic acid–buffered Listeria enrichment broth, University of Vermont medium–modified Listeria enrichment broth, and Fraser broth. The individual contributions of each selective agent in these media were also assessed, as well as the contribution of incubation temperature. Acriflavine hydrochloride and sodium nalidixate were ineffective at preventing the overgrowth of C. braakii; this resulted in substantially lower populations of Listeria than when the competitor was absent. At the higher levels, both of these selective agents were detrimental to Listeria populations. The highest enrichment populations of Listeria were observed when either NaCl or LiCl was present. In the absence of selective agents, the final populations of Listeria following competitive growth with C. braakii were not substantially affected by temperature; however, in the presence of selective agents, the Listeria populations were statistically higher at the higher incubation temperature. There are a limited number of selective agents available for use in Listeria-specific enrichment media, resulting in formulations that are only somewhat selective for this species. The optimization of current formulations may help researchers to improve Listeria recovery, particularly from products with a high microbial load. The understanding of the behavior and interactions between target and nontarget microorganisms in the presence of these available selective agents is a necessary step in the optimization of Listeria selective enrichment formulations.

Effect of Enrichment Media and Sampling Protocol on Recovery of Listeria monocytogenes

1990 ◽  
Vol 53 (6) ◽  
pp. 505-507 ◽  
Author(s):  
J. S. BAILEY ◽  
D. L. FLETCHER ◽  
N. A. COX
Keyword(s):  
Listeria Monocytogenes ◽  
Pure Culture ◽  
Sampling Protocol ◽  
Enrichment Broth ◽  
Secondary Enrichment ◽  
Pure Cultures ◽  
University Of Vermont

These studies examined the differences in recovery of Listeria monocytogenes from pure culture and in the populations of mixed aerobic microflora from chicken and Brie cheese incubated in University of Vermont (UVM) and Listeria enrichment broth (LEB) enrichment broths for different times and conditions. No significant differences were observed in levels of L. monocytogenes from pure cultures in UVM or LEB on any sampling day. No differences were observed in the levels of mixed microflora from Brie cheese in either UVM or LEB, but from chicken rinse the level of mixed flora competitors was significantly higher on all sampling days in LEB as compared to UVM. No differences were observed between a single enrichment in UVM or LEB for 2 d and a transfer to a secondary enrichment tube after 1 d. Overall, the level of mixed microflora capable of growing in enrichment broths was greater from chicken rinse than from Brie cheese. The ratio of L. monocytogenes to mixed microflora which survived the selective enrichments was most favorable for recovery of L. monocytogenes after 2 d of enrichment.

Comparison of EB and Fraser Enrichment Broths for the Detection of Listeria spp. and Listeria monocytogenes in Raw-Milk Dairy Products and Environmental Samples

1996 ◽  
Vol 59 (11) ◽  
pp. 1172-1175 ◽  
Author(s):  
GEERTRUI M. VLAEMYNCK ◽  
RENAAT MOERMANS
Keyword(s):  
Listeria Monocytogenes ◽  
Dairy Products ◽  
Raw Milk ◽  
Chain Reaction ◽  
Enrichment Broth ◽  
Selective Enrichment ◽  
Isolation Frequency ◽  
Detection And Identification ◽  
Significant Difference ◽  
Polymerase Chain

This study is a comparison of the isolation frequency of Listeria spp. and Listeria monocytogenes from selected naturally contaminated dairy products, especially soft smear-ripened cheeses from raw milk and samples of feces and rinse samples from the udder taken on the farm, by using an enrichment broth (EB) recommended by the International Dairy Federation and the U.S. Food and Drug Administration (IDF and FDA) or Fraser broth as the selective enrichment. Detection and identification were carried out according to the IDF protocols and a polymerase chain reaction technique. Listeria spp. were detected in 39.8% of the 570 samples while 15.3% were positive for L. monocytogenes. For cheese and curd samples, Fraser enrichment broth gave a statistically significant higher recovery for all Listeria spp. (26 to 21 %) as well as for L. monocytogenes in particular (9 to 1.4%). For raw milk and samples taken from feces and the udder rinse no significant difference was found between EB and Fraser broth. A combination of both enrichments resulted in an increase of recovery over all matrices by 15%.

Simultaneous Growth of Listeria monocytogenes and Listeria innocua

1993 ◽  
Vol 56 (7) ◽  
pp. 616-618 ◽  
Author(s):  
RUTH L. PETRAN ◽  
KATHERINE M. J. SWANSON
Keyword(s):  
Listeria Monocytogenes ◽  
Yeast Extract ◽  
Environmental Samples ◽  
Culture Media ◽  
Listeria Innocua ◽  
Enrichment Broth ◽  
University Of Vermont ◽  
Simultaneous Growth

Listeria spp. have been isolated from a wide variety of sources, and in many situations Listeria innocua is more commonly found than Listeria monocytogenes. Growth of three L. monocytogenes strains was studied when inoculated simultaneously with a rhamnose negative L. innocua strain into culture media and cheese sauce. Fraser broth (FB), Trypticase™ soy broth plus 0.6% yeast extract (TSB-YE), University of Vermont medium (UVM) modified Listeria enrichment broth, and cheese sauce were inoculated (ca. 102 cells per ml) and incubated for 24 h; FB, TSB-YE, and cheese sauce at 35°C, UVM at 30°C. Growth of four rhamnose-positive, L. innocua strains was also studied in culture media. Growth of L. monocytogenes was similar to that for L. innocua in TSB-YE or cheese sauce. However, in FB and UVM, L. innocua populations were significantly higher than L. monocytogenes. This occurred when media were inoculated individually or simultaneously. This may explain in part why L. innocua is isolated more frequently than L. monocytogenes from foods and environmental samples.

scholarly journals Bias in the Listeria monocytogenes Enrichment Procedure: Lineage 2 Strains Outcompete Lineage 1 Strains in University of Vermont Selective Enrichments

2005 ◽  
Vol 71 (2) ◽  
pp. 961-967 ◽  
Author(s):  
Jesper Bartholin Bruhn ◽  
Birte Fonnesbech Vogel ◽  
Lone Gram
Keyword(s):  
Growth Rate ◽  
Listeria Monocytogenes ◽  
Brain Heart Infusion ◽  
Selective Medium ◽  
Individual Growth ◽  
Selective Enrichment ◽  
Enrichment Procedure ◽  
True Representation ◽  
Lineage 2 ◽  
University Of Vermont

ABSTRACT Listeria monocytogenes can be isolated from a range of food products and may cause food-borne outbreaks or sporadic cases of listeriosis. L. monocytogenes is divided into three genetic lineages and 13 serotypes. Strains of three serotypes (1/2a, 1/2b, and 4b) are associated with most human cases of listeriosis. Of these, strains of serotypes 1/2b and 4b belong to lineage 1, whereas strains of serotype 1/2a and many other strains isolated from foods belong to lineage 2. L. monocytogenes is isolated from foods by selective enrichment procedures and from patients by nonselective methods. The aim of the present study was to investigate if the selective enrichment procedure results in a true representation of the subtypes of L. monocytogenes present in a sample. Eight L. monocytogenes strains (four lineage 1 strains and four lineage 2 strains) and one Listeria innocua strain grew with identical growth rates in the nonselective medium brain heart infusion (BHI), but differed in their growth rate in the selective medium University of Vermont medium I (UVM I). When coinoculated in UVM I, some strains completely outgrew other strains. This outcome was dependent on the lineage of L. monocytogenes rather than the individual growth rate of the strains. When inoculated at identical cell densities in UVM I, L. innocua outcompeted L. monocytogenes lineage 1 strains but not lineage 2 strains. In addition, lineage 2 L. monocytogenes strains outcompeted lineage 1 L. monocytogenes strains in all combinations tested, indicating a bias in strains selected by the enrichment procedures. Bias also occurred when coinoculating two lineage 2 or lineage 1 strains; however, it did not appear to correlate with origin (clinical versus food). Identical coinoculation experiments in BHI suggested that the selective compounds in UVM I and II influenced this bias. The results of the present study demonstrate that the selective procedures used for isolation of L. monocytogenes may not allow a true representation of the types present in foods. Our results could have a significant impact on epidemiological studies, as lineage 1 strains, which are often isolated from clinical cases of listeriosis, may be suppressed during enrichment by other L. monocytogenes lineages present in a food sample.

scholarly journals Detection and Isolation of Listeria monocytogenes from Food Samples: Implications of Sublethal Injury

2002 ◽  
Vol 85 (2) ◽  
pp. 495-500 ◽  
Author(s):  
Catherine W Donnelly
Keyword(s):  
Food Samples ◽  
Genetic Types ◽  
Selective Agents ◽  
Sample Enrichment ◽  
Sublethal Injury ◽  
Secondary Enrichment ◽  
Nutritional Needs ◽  
Low Levels ◽  
Meat Samples ◽  
University Of Vermont

Abstract Detection of L. monocytogenes is often limited by the performance of the enrichment media used to support bacterial growth to detectable levels. Because Listeria may exist at extremely low levels in foods, sample enrichment protocols must amplify these low initial populations to detectable limits. Listeria may also exist in an injured state in food products as a result of processing treatments such as heating, freezing, exposure to acids, or exposure to sanitizing compounds. Selective agents in enrichment media normally used for recovery of Listeria may inhibit repair and detection of sublethally injured Listeria, which may go on to repair, grow, and regain pathogenicity. Simple modifications to existing regulatory protocols, such as those that use more than one enrichment broth, raise sensitivity of detection to 90%. This review shows the efficacy of repair/enrichment strategies, which increase sensitivity of detection to 97.5–98.8% compared with 65–70% by standard regulatory protocols. Ribotype analysis of isolates obtained from meat samples reveals a complex microbial ecology, with striking differences in both number and distribution of distinct genetic types of Listeria, depending upon whether samples are enriched in selective or repair/enrichment media. In studies on enrichment of dairy environmental samples in University of Vermont medium and Listeria repair broth (UVM and LRB), combining these 2 primary enrichment media into a single tube of Fraser broth for dual secondary enrichment yielded a significantly higher percentage (p < 0.05) of Listeria-positive samples than did use of either LRB or UVM alone. Refinement of conventional Listeria recovery methods should consider the importance of the enrichment step, the nutritional needs of specific genetic types, and the physiological condition of Listeria isolates in foods.

scholarly journals SEL, a Selective Enrichment Broth for Simultaneous Growth of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes

2008 ◽  
Vol 74 (15) ◽  
pp. 4853-4866 ◽  
Author(s):  
Hyochin Kim ◽  
Arun K. Bhunia
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Salmonella Enterica ◽  
Single Experiment ◽  
Enrichment Broth ◽  
Selective Enrichment ◽  
E Coli ◽  
Multiplex Pcr Assay ◽  
Selective Enrichment Broth ◽  
Simultaneous Growth

ABSTRACT Multipathogen detection on a single-assay platform not only reduces the cost for testing but also provides data on the presence of pathogens in a single experiment. To achieve this detection, a multipathogen selective enrichment medium is essential to allow the concurrent growth of pathogens. SEL broth was formulated to allow the simultaneous growth of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes. The results were compared to those obtained with the respective individual selective enrichment broths, Rappaport-Vassiliadis (RV) for S. enterica, modified E. coli broth with 20 mg of novobiocin/liter for E. coli O157:H7, and Fraser broth for L. monocytogenes, and a currently used universal preenrichment broth (UPB). The growth of each pathogen in SEL inoculated at 101 or 103 CFU/ml was superior to that in the respective individual enrichment broth, except in the case of RV, in which Salmonella cells inoculated at both concentrations grew equally well. In mixed-culture experiments with cells of the three species present in equal concentrations or at a 1:10:1,000 ratio, the overall growth was proportional to the initial inoculation levels; however, the growth of L. monocytogenes was markedly suppressed when cells of this species were present at lower concentrations than those of the other two species. Further, SEL was able to resuscitate acid- and cold-stressed cells, and recovery was comparable to that in nonselective tryptic soy broth containing 6% yeast extract but superior to that in the respective individual selective broths. SEL promoted the growth of all three pathogens in a mixture in ready-to-eat salami and in turkey meat samples. Moreover, each pathogen was readily detected by a pathogen-specific immunochromatographic lateral-flow or multiplex PCR assay. Even though the growth of each pathogen in SEL was comparable to that in UPB, SEL inhibited greater numbers of nontarget organisms than did UPB. In summary, SEL was demonstrated to be a promising new multiplex selective enrichment broth for the detection of the three most prominent food-borne pathogens by antibody- or nucleic acid-based methods.

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