scholarly journals Validation of an ampicillin selection protocol to enrich for mutants of Listeria monocytogenes unable to replicate on fresh produce

2019 ◽  
Vol 366 (7) ◽  
Author(s):  
Victor Jayeola ◽  
C Parsons ◽  
L Gorski ◽  
S Kathariou
Keyword(s):  
Listeria Monocytogenes ◽  
Genetic Factors ◽  
Fresh Produce ◽  
Mixed Cultures ◽  
Bacterial Cells ◽  
Sensitive Mutant ◽  
Relative Recovery ◽  
Cold Sensitive ◽  
Selection Protocol

ABSTRACT Several outbreaks of listeriosis have implicated fresh produce but genetic factors required for growth of Listeria monocytogenes on produce remain poorly characterized. Based on the fact that β-lactam antibiotics only kill bacterial cells that are growing, we hypothesized that ampicillin selection can enrich for L. monocytogenes mutants unable to grow on produce. For validation, we examined relative recovery of L. monocytogenes strain 2011L-2858 and its cold-sensitive mutant L1E4 following inoculation of cantaloupe rind fragments with 1:1 mixture of the strains and incubation at 4°C with or without ampicillin. Listeria monocytogenes from rind fragments inoculated with the mixed cultures and incubated in the presence of ampicillin were used to inoculate fresh rind fragments for a second round of enrichment. In the presence of ampicillin, the proportion of L1E4 increased from 55% on day 0 to 78% on day 14, with higher recovery (85% after 14 days) in the second round of enrichment. These data suggested that L1E4 was enriched on cantaloupe rind fragments while growing cells of the wildtype were killed by ampicillin. Application of this protocol to transposon mutant libraries from three L. monocytogenes strains yielded several mutants unable to grow on cantaloupe. Thus, ampicillin selection can facilitate discovery of genes essential for growth of L. monocytogenes on fresh produce.

scholarly journals Changes in Listeria monocytogenes Membrane Fluidity in Response to Temperature Stress

2007 ◽  
Vol 73 (20) ◽  
pp. 6429-6435 ◽  
Author(s):  
Mohamed Badaoui Najjar ◽  
Michael Chikindas ◽  
Thomas J. Montville
Keyword(s):  
Listeria Monocytogenes ◽  
Membrane Fluidity ◽  
The Other ◽  
Sensitive Mutant ◽  
Homeoviscous Adaptation ◽  
Whole Cells ◽  
Food Borne ◽  
Cold Sensitive ◽  
The Difference ◽  
Response To Temperature

ABSTRACT Listeria monocytogenes is a food-borne pathogen that has been implicated in many outbreaks associated with ready-to-eat products. Listeria adjusts to various stresses by adjusting its membrane fluidity, increasing the uptake of osmoprotectants and cryoprotectants, and activating the σB stress factor. The present work examines the regulation of membrane fluidity through direct measurement based on fluorescent anisotropy. The membrane fluidities of L. monocytogenes Scott A, NR30, wt10403S, and cld1 cells cultured at 15 and 30°C were measured at 15 and 30°C. The membrane of the cold-sensitive mutant (cld1) was more rigid than the membranes of the other strains when grown at 30°C, but when grown at 15°C, it was able to adjust its membrane to approach the rigidity of the other strains. The difference in rigidities, as determined at 15 and 30°C, was greater in liposomes than in whole cells. The rates of fluidity adjustment and times required for whole cells to adjust to a different temperature were similar among strains but different from those of liposomes. This suggests that the cells had a mechanism for homeoviscous adaptation that was absent in liposomes.

Evaluation of biofilm-forming abilities of Listeria monocytogenes (ATCC 19115) and efficacy of different washing methods for removal of biofilm on apple

Food Research ◽  
2021 ◽  
Vol 5 (4) ◽  
pp. 259-265
Author(s):  
Y. Ali ◽  
H.Y. Mah ◽  
E.T. Phuah ◽  
S.N. Chen ◽  
S.K. Yeo ◽  
...  
Keyword(s):  
Supply Chain ◽  
Listeria Monocytogenes ◽  
Biofilm Formation ◽  
Time Course ◽  
Tap Water ◽  
Fresh Produce ◽  
Foodborne Illness ◽  
Food Supply Chain ◽  
Log Reduction ◽  
Crystal Violet Assay

Fresh produce can be contaminated at any stage along the food supply chain. In this study, apple was chosen to determine the time course of biofilm formation by Listeria monocytogenes (ATCC 19115), as well as to compare the efficacy of different household washing methods such as scrubbing with hands under running tap water, soaking with and without commercial vegetable wash with different treatment times in removing the biofilm formation by L. monocytogenes on apple surface. The biofilm formation was quantified using crystal violet assay and the result showed that L. monocytogenes took 18 hrs to form matured biofilm on apple surface. Besides, scrubbing apples with hands under running tap water for 30 s and 60 s were the most effective method which significantly removed (P<0.05) biofilm formed on the apple surface with approximately 5.93 log reduction. Soaking apples with vegetable wash for 5 mins and 10 mins were also found to be significantly effective (P<0.05) in reducing L. monocytogenes biofilm. Since L. monocytogenes can form matured biofilm on fresh produce, therefore efficient washing step is important before consuming fresh produce to lower the risk of foodborne illness.

Putative GTPase Gtr1p genetically interacts with the RanGTPase cycle in Saccharomyces cerevisiae

1996 ◽  
Vol 109 (9) ◽  
pp. 2311-2318 ◽  
Author(s):  
N. Nakashima ◽  
N. Hayashi ◽  
E. Noguchi ◽  
T. Nishimoto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Negative Regulator ◽  
Temperature Sensitive ◽  
Nucleotide Exchange ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Importin Alpha ◽  
Nucleotide Exchange Factor ◽  
Cold Sensitive ◽  
Gtpase Cycle

In order to identify a protein interacting with RCC1, a guanine nucleotide-exchange factor for the nuclear GTPase Ran, we isolated a series of cold-sensitive suppressors of mtr1-2, a temperature-sensitive mutant of the Saccharomyces cerevisiae RCC1 homologue. One of the isolated suppressor mutants was mutated in the putative GTPase Gtr1p, being designated as gtr1-11. It also suppressed other alleles of mtr1-2, srm1-1 and prp20-1 in contrast to overexpression of the S. cerevisiae Ran/TC4 homologue Gsp1p, previously reported to suppress prp20-1, but not mtr1-2 or srm1-1. Furthermore, gtr1-11 suppressed the rna1-1, temperature-sensitive mutant of the Gsp1p GTPase-activating protein, but not the srp1-31, temperature-sensitive mutant of the S. cerevisiae importin alpha homologue. mtr1-2, srm1-1 and prp20-1 were also suppressed by overexpression of the mutated Gtr1p, Gtr1-11p. In summary, Gtr1p that was localized in the cytoplasm by immunofluoresence staining was suggested to function as a negative regulator for the Ran/TC4 GTPase cycle.

A Solid Agar Overlay Method for Recovery of Heat-Injured Listeria monocytogenes

2006 ◽  
Vol 69 (2) ◽  
pp. 428-431 ◽  
Author(s):  
ZHINONG YAN ◽  
JOSHUA B. GURTLER ◽  
JEFFREY L. KORNACKI
Keyword(s):  
Listeria Monocytogenes ◽  
Yeast Extract ◽  
Foodborne Pathogens ◽  
Bacterial Cells ◽  
Heat Injury ◽  
Layer Method ◽  
Heat Treated ◽  
Solid Agar ◽  
Overlay Method ◽  
Agar Layer

A solid agar overlay method was developed for recovery of heat-injured Listeria monocytogenes. Presolidified nonselective tryptic soy agar with 0.6% yeast extract (TSAYE, 2% agar) was overlaid on top of solidified modified Oxford agar (MOX). Heat injury of L. monocytogenes was conducted at 58°C for 6 min in a jacketed flask filled with tryptic soy broth. Both noninjured and heat-treated L. monocytogenes cells were plated onto TSAYE, MOX, and TSAYE-MOX plates. No significant differences (P &gt; 0.05) in recovery were found among the three media for noninjured bacterial cells. Recovery of heat-injured L. monocytogenes cells on TSAYE-MOX overlay plates was equivalent to that on the nonselective TSAYE medium, whereas recovery on the selective MOX medium was significantly lower (P &lt; 0.05) compared with both TSAYE and the overlay plates. There were no significant differences (P &gt; 0.05) among the overlay plates prepared 0, 2, 4, 6, 8, 16, and 24 h prior to plating heat-injured bacterial cells. The TSAYE-MOX overlay also allowed differentiation of L. monocytogenes from a mixture of four other types of foodborne pathogens. This solid agar overlay method for recovery of heat-injured L. monocytogenes cells is less time-consuming and less complicated than the conventional overlay-underlay technique and the double overlay modification of the thin agar layer method and may allow for greater laboratory plating efficiencies.

scholarly journals Autonomous Growth of Isolated Single Listeria monocytogenes and Salmonella enterica Serovar Typhimurium Cells in the Absence of Growth Factors and Intercellular Contact

2010 ◽  
Vol 76 (8) ◽  
pp. 2600-2606 ◽  
Author(s):  
Barbara Roeder ◽  
Martin Wagner ◽  
Peter Rossmanith
Keyword(s):  
Growth Factors ◽  
Listeria Monocytogenes ◽  
Salmonella Enterica ◽  
Single Cells ◽  
Intercellular Contact ◽  
Bacterial Cells ◽  
Chi Square ◽  
Significant Difference ◽  
Serovar Typhimurium ◽  
Autonomous Growth

ABSTRACT The aim of this study was to observe growth of isolated single bacterial cells in the absence of growth factors and intercellular contact. In order to exclude stochastic uncertainties induced by dilution series, a new micromanipulation method was developed to ensure explicit results under visual control. This was performed with particular care for production of single prokaryotic cells and subsequent investigation of their autonomous growth. Over 450 single isolated Listeria monocytogenes and Salmonella enterica subsp. enterica serovar Typhimurium cells in lag, log, and stationary growth phases were investigated by this method, which included thoroughly washing the cells. The proportion of living cells within the initial cultures was compared to the proportion of positive samples after enrichment of the separated single cells. This resulted in P values of ≥0.05 using the chi-square test for statistical analysis, indicating no significant difference, and clearly demonstrates reproduction of isolated single bacterial cells without the need for growth factors or intercellular contact. Ease of handling of the apparatus and good performance of the cleaning procedures were achieved, as was validation of the method, demonstrating its suitability for routine laboratory use.

scholarly journals Viable-but-NonculturableListeria monocytogenesandSalmonella entericaSerovar Thompson Induced by Chlorine Stress Remain Infectious

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
pp. e00540-18 ◽  
Author(s):  
Callum J. Highmore ◽  
Jennifer C. Warner ◽  
Steve D. Rothwell ◽  
Sandra A. Wilks ◽  
C. William Keevil
Keyword(s):  
Listeria Monocytogenes ◽  
Caenorhabditis Elegans ◽  
Life Span ◽  
Fresh Produce ◽  
Environmental Stresses ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Content Type ◽  
Food Borne Pathogens ◽  
Food Borne

ABSTRACTThe microbiological safety of fresh produce is monitored almost exclusively by culture-based detection methods. However, bacterial food-borne pathogens are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses such as chlorine, which is commonly used for fresh produce decontamination. Here, complete VBNC induction of green fluorescent protein-taggedListeria monocytogenesandSalmonella entericaserovar Thompson was achieved by exposure to 12 and 3 ppm chlorine, respectively. The pathogens were subjected to chlorine washing following incubation on spinach leaves. Culture data revealed that total viableL. monocytogenesandSalmonellaThompson populations became VBNC by 50 and 100 ppm chlorine, respectively, while enumeration by direct viable counting found that chlorine caused a <1-log reduction in viability. The pathogenicity of chlorine-induced VBNCL. monocytogenesandSalmonellaThompson was assessed by usingCaenorhabditis elegans. Ingestion of VBNC pathogens byC. elegansresulted in a significant life span reduction (P= 0.0064 andP< 0.0001), and no significant difference between the life span reductions caused by the VBNC and culturableL. monocytogenestreatments was observed.L. monocytogeneswas visualized beyond the nematode intestinal lumen, indicating resuscitation and cell invasion. These data emphasize the risk that VBNC food-borne pathogens could pose to public health should they continue to go undetected.IMPORTANCEMany bacteria are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses. VBNC cells cannot be detected by standard laboratory culture techniques, presenting a problem for the food industry, which uses these techniques to detect pathogen contaminants. This study found that chlorine, a sanitizer commonly used for fresh produce, induces a VBNC state in the food-borne pathogensListeria monocytogenesandSalmonella enterica. It was also found that chlorine is ineffective at killing total populations of the pathogens. A life span reduction was observed inCaenorhabditis elegansthat ingested these VBNC pathogens, with VBNCL. monocytogenesas infectious as its culturable counterpart. These data show that VBNC food-borne pathogens can both be generated and avoid detection by industrial practices while potentially retaining the ability to cause disease.

scholarly journals Modeling Stochastic Variability in the Numbers of Surviving Salmonella enterica, Enterohemorrhagic Escherichia coli, and Listeria monocytogenes Cells at the Single-Cell Level in a Desiccated Environment

2016 ◽  
Vol 83 (4) ◽  
Author(s):  
Kento Koyama ◽  
Hidekazu Hokunan ◽  
Mayumi Hasegawa ◽  
Shuso Kawamura ◽  
Shigenobu Koseki
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Probability Distribution ◽  
Poisson Process ◽  
Poisson Distribution ◽  
Salmonella Enterica ◽  
Bacterial Cells ◽  
Content Type ◽  
Cell Numbers ◽  
Inactivation Process

ABSTRACT Despite effective inactivation procedures, small numbers of bacterial cells may still remain in food samples. The risk that bacteria will survive these procedures has not been estimated precisely because deterministic models cannot be used to describe the uncertain behavior of bacterial populations. We used the Poisson distribution as a representative probability distribution to estimate the variability in bacterial numbers during the inactivation process. Strains of four serotypes of Salmonella enterica, three serotypes of enterohemorrhagic Escherichia coli, and one serotype of Listeria monocytogenes were evaluated for survival. We prepared bacterial cell numbers following a Poisson distribution (indicated by the parameter λ, which was equal to 2) and plated the cells in 96-well microplates, which were stored in a desiccated environment at 10% to 20% relative humidity and at 5, 15, and 25°C. The survival or death of the bacterial cells in each well was confirmed by adding tryptic soy broth as an enrichment culture. Changes in the Poisson distribution parameter during the inactivation process, which represent the variability in the numbers of surviving bacteria, were described by nonlinear regression with an exponential function based on a Weibull distribution. We also examined random changes in the number of surviving bacteria using a random number generator and computer simulations to determine whether the number of surviving bacteria followed a Poisson distribution during the bacterial death process by use of the Poisson process. For small initial cell numbers, more than 80% of the simulated distributions (λ = 2 or 10) followed a Poisson distribution. The results demonstrate that variability in the number of surviving bacteria can be described as a Poisson distribution by use of the model developed by use of the Poisson process. IMPORTANCE We developed a model to enable the quantitative assessment of bacterial survivors of inactivation procedures because the presence of even one bacterium can cause foodborne disease. The results demonstrate that the variability in the numbers of surviving bacteria was described as a Poisson distribution by use of the model developed by use of the Poisson process. Description of the number of surviving bacteria as a probability distribution rather than as the point estimates used in a deterministic approach can provide a more realistic estimation of risk. The probability model should be useful for estimating the quantitative risk of bacterial survival during inactivation.

Two Generally Recognized as Safe Surfactants plus Acidulants Inactivate Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in Suspension or on Dip-Inoculated Grape Tomatoes

2020 ◽  
Vol 83 (4) ◽  
pp. 637-643
Author(s):  
JOSHUA B. GURTLER
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Fresh Produce ◽  
Free Chlorine ◽  
Sodium Dodecylbenzene Sulfonate ◽  
Escherichia Coli O157 ◽  
E Coli

ABSTRACT Contamination of fresh produce with the foodborne pathogens Salmonella enterica, Listeria monocytogenes, and Escherichia coli O157:H7 continues to be problematic, resulting in outbreaks of foodborne illness and costly corporate recalls. Various individual concentrations of citric or lactic acids (0.35 to 0.61%) or isopropyl citrate (0.16 to 0.54%) combined with two generally recognized as safe surfactants, 0.025% sodium-2-ethyl-hexyl sulfate and 0.025% sodium dodecylbenzene-sulfonate, were tested against these three pathogens in suspension and when inoculated and dried on the surface of grape tomatoes. The efficacy of sodium hypochlorite (NaClO; at 46 ppm) was also evaluated under dirty and clean conditions in suspension after addition of 0.3 or 0.03% bovine serum albumin, respectively, as an organic load. NaClO (46 ppm) inactivated the three pathogens in suspension by &lt;0.76 log CFU/mL after 5 min in the presence of 0.3% bovine serum albumin, whereas 9 and 15 ppm of free chlorine inactivated the pathogens by 0.64 and 2.77 log CFU/mL, respectively, after 5 min under clean conditions. Isopropyl citrate (0.16% acidulant) plus 0.05% total concentration of the two surfactants inactivated the pathogens in suspension by up to 7.0 log CFU/mL within 2 min. When applied to grape tomatoes for 2 min, 0.54% isopropyl citrate plus 0.025% concentrations of each of the two surfactants reduced Salmonella, E. coli O157:H7, and L. monocytogenes by as much as ca. 5.47, 4.89, and 4.19 log CFU/g, respectively. These reductions were significantly greater than those achieved with 49 ppm of free chlorine. Citric acid and lactic acid plus surfactant washes achieved greater inactivation than water-only washes, reducing Salmonella, E. coli O157:H7, and L. monocytogenes on tomatoes by up to 4.90, 4.37, and 3.98 log CFU/g, respectively. These results suggest that these combinations of acidulants and surfactants may be an effective tool for preventing cross-contamination during the washing of grape tomatoes, for reducing pathogens on the fruit itself, and as an alternative to chlorine for washing fresh produce. HIGHLIGHTS

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