scholarly journals Optimized, One-Step, Recovery-Enrichment Broth for Enhanced Detection of Listeria monocytogenes in Pasteurized Milk and Hot Dogs

2002 ◽  
Vol 85 (2) ◽  
pp. 501-504 ◽  
Author(s):  
Stephen J Knabel
Keyword(s):  
Listeria Monocytogenes ◽  
False Negative ◽  
Molecular Methods ◽  
Vegetative Cells ◽  
Enrichment Broth ◽  
Penn State ◽  
Bacillus Spp ◽  
One Step ◽  
Bacillus Spores ◽  
Low Levels

Abstract A one-step, recovery-enrichment broth, optimized Penn State University (oPSU) broth, was developed to consistently detect low levels of injured and uninjured Listeria monocytogenes cells in ready-to-eat foods. The oPSU broth contains special selective agents that inhibit growth of background flora without inhibiting recovery of injured Listeria cells. After recovery in the anaerobic section of oPSU broth, Listeria cells migrated to the surface, forming a black zone. This migration separated viable from nonviable cells and the food matrix, thereby reducing inhibitors that prevent detection by molecular methods. The high Listeria-to-background ratio in the black zone resulted in consistent detection of low levels of L. monocytogenes in pasteurized foods by both cultural and molecular methods, and greatly reduced both false-negative and false-positive results. oPSU broth does not require transfer to a secondary enrichment broth, making it less laborious and less subject to external contamination than 2-step enrichment protocols. Addition of 150mM d-serine prevented germination of Bacillus spores, but not the growth of vegetative cells. Replacement of d-serine with 12 mg/L acriflavin inhibited growth of vegetative cells of Bacillus spp. without inhibiting recovery of injured Listeria cells. oPSU broth may allow consistent detection of low levels of injured and uninjured cells of L. monocytogenes in pasteurized foods containing various background microflora.

DETECTION AND DIFFERENTIATION OF SPORE AND VEGETATIVE FORMS OF BACILLUS SPP. USING INFRARED SPECTROSCOPIC METHODS

2008 ◽  
Vol 18 (02) ◽  
pp. 417-427
Author(s):  
DIANE ST. AMANT ◽  
MARK CAMPBELL ◽  
ANDREW BECK ◽  
LESLIE WILLIAMS ◽  
JENNIFER MINTER ◽  
...  
Keyword(s):  
Thin Film ◽  
Infrared Spectroscopy ◽  
Attenuated Total Reflection ◽  
Bacillus Species ◽  
Infrared Spectrometry ◽  
Limited Information ◽  
Transmission Method ◽  
Vegetative Cells ◽  
Bacillus Spp ◽  
Bacillus Spores

Infrared spectroscopy has been demonstrated as a powerful tool for taxonomic classification of bacteria when the microbes are grown and sampled under carefully controlled conditions. Infrared spectroscopy affords limited information about relative proportions of certain chemical functional groups in whole microbial cells. The objective of this work is to elucidate the ability of infrared spectroscopy to identify and speciate Bacillus spp. regardless of sample history. Spectrometers utilize different scanning methods to collect infrared absorption spectra. We employed three; transmission through a thin film, transmission infrared microscopy, and Attenuated Total Reflection (ATR). Target organisms include Bacillus anthracis, and several near neighbors. Each strain was cultured at 24°C and 35°C on three solid media. Microorganisms were incubated for up to ten days to include vegetative cells, spore formation and mature spores. Triplicate microbe samples were prepared and analyzed according to instrument requirements using the three measurement modes. Triplicate samples of BSL-3 organisms were analyzed only by the thin film transmission method. Spectral data was analyzed using the cluster analysis function of OPUS software. We report that infrared spectrometry is capable of discerning Bacillus spores from vegetative cells and the phylogenic clustering of Bacillus species according to pathogenicity levels via infrared spectral analysis.

scholarly journals Influence of Stress on Single-Cell Lag Time and Growth Probability for Listeria monocytogenes in Half Fraser Broth

2009 ◽  
Vol 75 (10) ◽  
pp. 3069-3076 ◽  
Author(s):  
Claire Dupont ◽  
Jean-Christophe Augustin
Keyword(s):  
Listeria Monocytogenes ◽  
Single Cell ◽  
Physiological State ◽  
False Negative ◽  
Lag Time ◽  
Enrichment Broth ◽  
A Cell ◽  
Lag Times ◽  
The Impact ◽  
Growth Probability

ABSTRACT The impacts of 12 common food industry stresses on the single-cell growth probability and single-cell lag time distribution of Listeria monocytogenes were determined in half Fraser broth, the primary enrichment broth of the International Organization for Standardization detection method. First, it was determined that the ability of a cell to multiply in half Fraser broth is conditioned by its history (the probability for a cell to multiply can be decreased to 0.05), meaning that, depending on the stress in question, the risk of false-negative samples can be very high. Second, it was established that when cells are injured, the single-cell lag times increase in mean and in variability and that this increase represents a true risk of not reaching the detection threshold of the method in the enrichment broth. No relationship was observed between the impact on single-cell lag times and that on growth probabilities. These results emphasize the importance of taking into account the physiological state of the cells when evaluating the performance of methods to detect pathogens in food.

scholarly journals Postenrichment Population Differentials Using Buffered Listeria Enrichment Broth: Implications of the Presence of Listeria innocua on Listeria monocytogenes in Food Test Samples†

2013 ◽  
Vol 76 (11) ◽  
pp. 1854-1862 ◽  
Author(s):  
ASHLEY L. KEYS ◽  
RACHEL C. DAILEY ◽  
ANTHONY D. HITCHINS ◽  
R. DERIKE SMILEY
Keyword(s):  
Growth Rate ◽  
Listeria Monocytogenes ◽  
Listeria Innocua ◽  
Population Differences ◽  
Enrichment Broth ◽  
Listeria Species ◽  
Plating Method ◽  
Low Levels ◽  
Pathogen Recovery ◽  
Enrichment Step

The recovery of low levels of Listeria monocytogenes from foods is complicated by the presence of competing microorganisms. Nonpathogenic species of Listeria pose a particular problem because variation in growth rate during the enrichment step can produce more colonies of these nontarget cells on selective and/or differential media, resulting in a preferential recovery of nonpathogens, especially Listeria innocua. To gauge the extent of this statistical barrier to pathogen recovery, 10 isolates each of L. monocytogenes and L. innocua were propagated together from approximately equal initial levels using the current U.S. Food and Drug Administration's enrichment procedure. In the 100 isolate pairs, an average 1.3-log decrease was found in the 48-h enrichment L. monocytogenes population when L. innocua was present. In 98 of the 100 isolate pairs, L. innocua reached higher levels at 48 h than did L. monocytogenes, with a difference of 0.2 to 2.4 log CFU/ml. The significance of these population differences was apparent by an increase in the difficulty of isolating L. monocytogenes by the streak plating method. L. monocytogenes went completely undetected in 18 of 30 enrichment cultures even after colony isolation was attempted on Oxoid chromogenic Listeria agar. This finding suggests that although both Listeria species were present on the plate, the population differential between them restricted L. monocytogenes to areas of the plate with confluent growth and that isolated individual colonies were only L. innocua.

Three Clusters of Bacillus Pseudobacteremia Related to a Radiometric Blood Culture Analyzer

1984 ◽  
Vol 5 (2) ◽  
pp. 71-74 ◽  
Author(s):  
Inge Gurevich ◽  
Patricia Tafuro ◽  
Sharon P. Krystofiak ◽  
Robert D. Kalter ◽  
Burke A. Cunha
Keyword(s):  
Blood Culture ◽  
Clinical Signs ◽  
Blood Cultures ◽  
Bacillus Species ◽  
Common Source ◽  
Air Conditioner ◽  
Close Proximity ◽  
Bacillus Spp ◽  
Multiple Blood ◽  
Bacillus Spores

AbstractDuring a ten-month period from September 1981 to July 1982 three episodes of pseudobacteremia due to Bacillus species occurred at this 550-bed institution. The first involved eight isolates, the second 11, and the third seven isolates of the organism, all with the same antibiogram.The patients involved did not exhibit clinical signs of septicemia, and in only one case was more than one specimen per patient positive when multiple blood samples were obtained. Occasional blood cultures of Bacillus species identified in between clusters revealed a different antibiogram.Extensive epidemiologic investigation of patient locations, phlebotomists, and time of cultures yielded no common source. Components involved in the transport and processing of blood cultures, including the radiometric blood culture processor, were also sampled but without recovery of the organism. After the last episode, a layer of dust was noted inside the machine, and culture of this dust grew Bacillus spp. with the same antibiogram as those found in the blood cultures. The filter from an air conditioning unit in close proximity to the machine grew several species of Bacillus.It is presumed that Bacillus spores in the dust were introduced into the blood culture bottles following the heat sterilization of the gas sampling (inoculation/removal) needles.Modification of the cover of the machine was undertaken to prevent access of dust bearing microbes to the inside of the machine. In addition, maintenance now includes regular disinfection/cleaning of the “floor” of the machine, and more frequent changes of the air conditioner filter.

scholarly journals The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria, and the localization of vasodilator-stimulated phosphoprotein and profilin.

1996 ◽  
Vol 135 (3) ◽  
pp. 647-660 ◽  
Author(s):  
G A Smith ◽  
J A Theriot ◽  
D A Portnoy
Keyword(s):  
Listeria Monocytogenes ◽  
Actin Polymerization ◽  
Consensus Sequence ◽  
Wild Type ◽  
Movement Rate ◽  
Bacterial Surface ◽  
Rate Dependent ◽  
Repeat Domain ◽  
Low Levels ◽  
Rate Of Movement

The ActA protein is responsible for the actin-based movement of Listeria monocytogenes in the cytosol of eukaryotic cells. Analysis of mutants in which we varied the number of proline-rich repeats (PRR; consensus sequence DFPPPPTDEEL) revealed a linear relationship between the number of PRRs and the rate of movement, with each repeat contributing approximately 2-3 microns/min. Mutants lacking all functional PRRs (generated by deletion or point mutation) moved at rates 30% of wild-type. Indirect immunofluorescence indicated that the PRRs were directly responsible for binding of vasodilator-stimulated phosphoprotein (VASP) and for the localization of profilin at the bacterial surface. The long repeats, which are interdigitated between the PRRs, increased the frequency with which actin-based motility occurred by a mechanism independent of the PRRs, VASP, and profilin. Lastly, a mutant which expressed low levels of ActA exhibited a phenotype indicative of a threshold; there was a very low percentage of moving bacteria, but when movement did occur, it was at wild-type rates. These results indicate that the ActA protein directs at least three separable events: (1) initiation of actin polymerization that is independent of the repeat region; (2) initiation of movement dependent on the long repeats and the amount of ActA; and (3) movement rate dependent on the PRRs.

Detection of Bacillus Spores Using PCR and FTA Filters

2004 ◽  
Vol 67 (5) ◽  
pp. 1036-1038 ◽  
Author(s):  
KEITH A. LAMPEL ◽  
DEANNE DYER ◽  
LEROY KORNEGAY ◽  
PALMER A. ORLANDI
Keyword(s):  
Nested Pcr ◽  
Pcr Primers ◽  
Food Samples ◽  
Microbial Pathogens ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Conserved Sequence ◽  
Bacillus Spp ◽  
Bacillus Spores ◽  
Template Dna

Emphasis has been placed on developing and implementing rapid detection systems for microbial pathogens. We have explored the utility of expanding FTA filter technology for the preparation of template DNA for PCR from bacterial spores. Isolated spores from several Bacillus spp., B. subtilis, B. cereus, and B. megaterium, were applied to FTA filters, and specific DNA products were amplified by PCR. Spore preparations were examined microscopically to ensure that the presence of vegetative cells, if any, did not yield misleading results. PCR primers SRM86 and SRM87 targeted a conserved region of bacterial rRNA genes, whereas primers Bsub5F and Bsub3R amplified a product from a conserved sequence of the B. subtilis rRNA gene. With the use of the latter set of primers for nested PCR, the sensitivity of the PCR-based assay was increased. Overall, 53 spores could be detected after the first round of PCR, and the sensitivity was increased to five spores by nested PCR. FTA filters are an excellent platform to remove PCR inhibitors and have universal applications for environmental, clinical, and food samples.

scholarly journals Evaluation of sanitary quality of goat milk in dairy industries from the Cariri region, state of Paraíba

2021 ◽  
Vol 10 (12) ◽  
pp. e596101220735
Author(s):  
Iara Nunes de Siqueira ◽  
Aline Antas Cordeiro Cavalcanti ◽  
Joyce Galvão de Souza ◽  
Filipe Jordão Pereira de Medeiros ◽  
João Carlos Taveira ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Pathogenic Bacteria ◽  
Goat Milk ◽  
Salmonella Spp ◽  
Pasteurized Milk ◽  
Enterococcus Spp ◽  
Bacillus Spp ◽  
Thermotolerant Coliforms ◽  
Staphylococcus Spp

The sanitary evaluation of equipment and hands is fundamental to investigate the presence of pathogens in the dairy industry. Then, this study aims to evaluate the sanitization of equipment, workers’ hands, raw and pasteurized milk in goat milk dairies in the Cariri region, state of Paraíba.  Collected 32 samples of four dairies represented by letters A, B, C, and D. The followings contents were analyzed: mesophiles, total and thermotolerant coliforms, Escherichia coli, Staphylococcus aureus, Samonella spp. and Listeria monocytogenes in the reception tank, pasteurization tank, packing machine, package, wall, workers’ hand, and each dairy’s raw and pasteurized milk. After isolation, 84 colonies were confirmed by MALDI TOF. The indicator microorganisms presented variations for the workers’ hands, while A and B stayed within the patterns. For the equipment, only dairy B was within limits. They were out of the standard for mesophiles, total coliforms, and thermotolerant regarding raw and pasteurized milk. The microorganisms, the Enterobacteriaceae family presented a higher frequency, with 77.38%, and within this family, Escherichia coli, Klebsiella spp., and Enterobacter spp. were the most prevalent. Gram-positive corresponded to 22.62%, Bacillus spp., Staphylococcus spp., Enterococcus spp., and Macrococcus caseolyticus. Listeria monocytogenes and Salmonella spp. were not isolated. These demonstrate failures in goat milk processing with pathogenic bacteria in several dairy plants, indicating the need to adjust the product’s quality control.

Comparison of methods for optimum detection of stressed and low levels of Listeria monocytogenes

1992 ◽  
Vol 9 (2) ◽  
pp. 127-145 ◽  
Author(s):  
Donald W. Warburton ◽  
Jeffrey M. Farber ◽  
Conrad Powell ◽  
Narayan P. Tiwari ◽  
Susan Read ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Comparison Of Methods ◽  
Optimum Detection ◽  
Low Levels

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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