scholarly journals Elucidating Escherichia Coli O157:H7 Colonization and Internalization in Cucumbers Using an Inverted Fluorescence Microscope and Hyperspectral Microscopy

2019 ◽  
Vol 7 (11) ◽  
pp. 499
Author(s):  
Yeting Sun ◽  
Dan Wang ◽  
Yue Ma ◽  
Hongyang Guan ◽  
Hao Liang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fluorescence Microscope ◽  
Vascular Bundles ◽  
Escherichia Coli O157 ◽  
Bacterial Cells ◽  
Plant Interactions ◽  
Flower Bud ◽  
Cucumis Sativus L ◽  
Spectral Angle Mapper ◽  
E Coli

Contamination of fresh cucumbers (Cucumis sativus L.) with Escherichia coli O157:H7 can impact the health of consumers. Despite this, the pertinent mechanisms underlying E. coli O157:H7 colonization and internalization remain poorly documented. Herein we aimed to elucidate these mechanisms in cucumbers using an inverted fluorescence microscope and hyperspectral microscopy. We observed that E. coli O157:H7 primarily colonized around the stomata on cucumber epidermis without invading the internal tissues of intact cucumbers. Once the bacterial cells had infiltrated into the internal tissues, they colonized the cucumber placenta and vascular bundles (xylem vessels, in particular), and also migrated along the xylem vessels. Moreover, the movement rate of E. coli O157:H7 from the stalk to the flower bud was faster than that from the flower bud to the stalk. We then used hyperspectral microscope imaging to categorize the infiltrated and uninfiltrated areas with high accuracy using the spectral angle mapper (SAM) classification method, which confirmed the results obtained upon using the inverted fluorescence microscope. We believe that our results are pivotal for developing science-based food safety practices, interventions for controlling E. coli O157:H7 internalization, and new methods for detecting E. coli O157:H7-plant interactions.

Attachment of Escherichia coli O157:H7 and Other Bacterial Cells Grown in Two Media to Beef Adipose and Muscle Tissues

1997 ◽  
Vol 60 (2) ◽  
pp. 102-106 ◽  
Author(s):  
LAURA CABEDO ◽  
JOHN N. SOFOS ◽  
GLENN R. SCHMIDT ◽  
GARY C. SMITH
Keyword(s):  
Escherichia Coli ◽  
Adipose Tissue ◽  
Muscle Tissue ◽  
Storage Period ◽  
Escherichia Coli O157 ◽  
Bacterial Cells ◽  
Liquid Droplets ◽  
E Coli ◽  
Attachment Strength ◽  
Beef Muscle

Three strains of Escherichia coli O157:H7 were grown in tryptic soy broth (TSB) or in a sterile cattle manure extract at 35°C for 18 ± 2 h. Aliquots from both inocula containing 106 CFU/ml were used to inoculate 1-cm3 cubes of beef muscle or adipose tissue by immersion for 20 min at 21°C. After removal from the inoculum, one-half of the samples were analyzed for bacterial cell numbers and pH, and the other half were stored at 4°C for 2 or 3 h before analysis. Samples were analyzed by enumerating bacteria present in liquid droplets deposited on the tissue and bacteria loosely or strongly attached to the tissue in order to determine attachment strength. Total numbers of cells on beef muscle tissue (bacteria in liquid droplets, as well as those loosely and strongly attached) were 5.65 ± 0.14 and 5.76 ± 0.26 log CFU/cm2 for E. coli O157:H7 inocula grown in TSB and manure extract, respectively. The differences in attachment strength between inocula from the two media were not significant (P > 0.05). A 2-h storage period after exposure of muscle tissue to an E. coli O157:H7 inoculum did not influence attachment strength. Numbers of bacteria attached to adipose tissue and muscle (5.31 ± 0.08 and 5.48 ± 0.09 log CFU/cm2, respectively) were not significantly different (P > 0.05). After 3 h at 4°C, the attachment strength of E. coli O157:H7 cells on muscle or adipose tissue had not changed. Overall, the culture medium and type of beef tissue did not affect the numbers of E. coli O157:H7 cells attached, nor the strength of their attachment, to muscle or adipose tissue.

Effectiveness and Functional Mechanism of a Multicomponent Sanitizer against Biofilms Formed by Escherichia coli O157:H7 and Five Salmonella Serotypes Prevalent in the Meat Industry

2020 ◽  
Vol 83 (4) ◽  
pp. 568-575
Author(s):  
RONG WANG ◽  
YOU ZHOU ◽  
NORASAK KALCHAYANAND ◽  
DAYNA M. HARHAY ◽  
TOMMY L. WHEELER
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Three Dimensional ◽  
Short Exposure ◽  
Escherichia Coli O157 ◽  
Bacterial Cells ◽  
Meat Processing ◽  
E Coli ◽  
Biofilm Removal ◽  
Biofilm Structure

ABSTRACT Biofilm formation by Escherichia coli O157:H7 and Salmonella enterica at meat processing plants poses a potential risk of meat product contamination. Many common sanitizers are unable to completely eradicate biofilms formed by these foodborne pathogens because of the three-dimensional biofilm structure and the presence of bacterial extracellular polymeric substances (EPSs). A novel multifaceted approach combining multiple chemical reagents with various functional mechanisms was used to enhance the effectiveness of biofilm control. We tested a multicomponent sanitizer consisting of a quaternary ammonium compound (QAC), hydrogen peroxide, and the accelerator diacetin for its effectiveness in inactivating and removing Escherichia coli O157:H7 and Salmonella enterica biofilms under meat processing conditions. E. coli O157:H7 and Salmonella biofilms on common contact surfaces were treated with 10, 20, or 100% concentrations of the multicomponent sanitizer solution for 10 min, 1 h, or 6 h, and log reductions in biofilm mass were measured. Scanning electron microscopy (SEM) was used to directly observe the effect of sanitizer treatment on biofilm removal and bacterial morphology. After treatment with the multicomponent sanitizer, viable E. coli O157:H7 and Salmonella biofilm cells were below the limit of detection, and the prevalence of both pathogens was low. After treatment with a QAC-based control sanitizer, surviving bacterial cells were countable, and pathogen prevalence was higher. SEM analysis of water-treated control samples revealed the three-dimensional biofilm structure with a strong EPS matrix connecting bacteria and the contact surface. Treatment with 20% multicomponent sanitizer for 10 min significantly reduced biofilm mass and weakened the EPS connection. The majority of the bacterial cells had altered morphology and compromised membrane integrity. Treatment with 100% multicomponent sanitizer for 10 min dissolved the EPS matrix, and no intact biofilm structure was observed; instead, scattered clusters of bacterial aggregates were detected, indicating the loss of cell viability and biofilm removal. These results indicate that the multicomponent sanitizer is effective, even after short exposure with dilute concentrations, against E. coli O157:H7 and S. enterica biofilms. HIGHLIGHTS

Primers with 5′ Flaps Improve the Efficiency and Sensitivity of Multiplex PCR Assays for the Detection of Salmonella and Escherichia coli O157:H7

2013 ◽  
Vol 76 (4) ◽  
pp. 668-673 ◽  
Author(s):  
CHRIS TIMMONS ◽  
SHEFALI DOBHAL ◽  
JACQUELINE FLETCHER ◽  
LI MARIA MA
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Fold Increase ◽  
Detection Sensitivity ◽  
Escherichia Coli O157 ◽  
Bacterial Cells ◽  
Robust Detection ◽  
E Coli ◽  
Pcr Multiplex ◽  
Pcr Assays

Foodborne illnesses caused by Salmonella enterica and Escherichia coli O157:H7 are worldwide health concerns. Rapid, sensitive, and robust detection of these pathogens in foods and in clinical and environmental samples is essential for routine food quality testing, effective surveillance, and outbreak investigations. The aim of this study was to evaluate the effect on PCR sensitivity of adding a short, AT-rich overhanging nucleotide sequence (flap) to the 5′ end of PCR primers specific for the detection of Salmonella and E. coli O157:H7. Primers targeting the invA gene of Salmonella and the rfbE gene of E. coli O157:H7 were synthesized with or without a 12-bp, AT-rich 5′ flap (5′-AATAAATCATAA-3′). Singleplex PCR, multiplex PCR, and real-time PCR sensitivity assays were conducted using purified bacterial genomic DNA and crude cell lysates of bacterial cells. The effect of background flora on detection was evaluated by spiking tomato and jalapeno pepper surface washes with E. coli O157:H7 and Salmonella Saintpaul. When targeting individual pathogens, end-point PCR assays using flap-amended primers were more efficient than nonamended primers, with 20.4 and 23.5% increases in amplicon yield for Salmonella and E. coli O157:H7, respectively. In multiplex PCR assays, a 10- to 100-fold increase in detection sensitivity was observed when the primer flap sequence was incorporated. This improvement in both singleplex and multiplex PCR efficiency and sensitivity can lead to improved Salmonella and E. coli O157:H7 detection.

Effects of Oxidative Compounds on Thermotolerance in Escherichia coli O157:H7 Strains EO139 and 380-94

2005 ◽  
Vol 68 (11) ◽  
pp. 2443-2446 ◽  
Author(s):  
ISABEL C. BLACKMAN ◽  
YOUNG W. PARK ◽  
MARK A. HARRISON
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Ascorbic Acid ◽  
Lethal Effect ◽  
Iron Chloride ◽  
Plate Count ◽  
Escherichia Coli O157 ◽  
Bacterial Cells ◽  
E Coli ◽  
Chloride Hexahydrate

An oxidative complex composed of ferric iron chloride hexahydrate, ADP, and ascorbic acid can generate hydrogen peroxide and hydroxyl radicals in fibroblasts. These compounds are naturally found in meat and meat-based products and may elicit oxidative stress on Escherichia coli O157:H7, thus conferring thermotolerance to the bacterium due to the phenomenon of the global stress response. The effect of the levels of the oxidative complex on the thermotolerance of E. coli O157:H7 was investigated. Cultures of E. coli O157:H7 strains EO139 and 380-94 were mixed in three different concentrations (10:10: 40, 15:15:60, and 20:20:80 μM) of the oxidative complex (iron III chloride, ADP, and ascorbic acid, respectively). The samples were inserted into capillary tubes and heated in a circulating water bath at 59 and 60°C for EO139 and 380-94, respectively. Tubes were removed at intervals of 5 min for up to 1 h and contents spirally plated on plate count agar that was incubated for 48 h at 37°C. The thermotolerance of both E. coli O157:H7 strains EO139 and 380-94 was influenced by the concentrations of the oxidative complex. The ratio of 10:10:40 μM enhanced thermotolerance of EO139 and 390-94 at 59 and 60°C, respectively. However, exposure to the ratios of 15:15:60 and 20:20:80 μM rendered the pathogen more sensitive to the lethal effect and did not enhance the thermotolerance of the cells. The significance of this study is twofold. This experiment proves that oxidative stress can enhance thermotolerance of bacterial cells induced by an oxidative complex if only in a specific ratio and concentration. It is possible to speculate that if the chemical compounds are present in this ratio in meats, they may enhance the thermal resistance of E. coli O157:H7 and make the bacteria more difficult to eliminate, thus increasing the risk of foodborne illness in consumers.

Identification of the Cellular Location of Internalized Escherichia coli O157:H7 in Mung Bean, Vigna radiata, by Immunocytochemical Techniques

2011 ◽  
Vol 74 (8) ◽  
pp. 1224-1230 ◽  
Author(s):  
AMANDA J. DEERING ◽  
ROBERT E. PRUITT ◽  
LISA J. MAUER ◽  
BRADLEY L. REUHS
Keyword(s):  
Escherichia Coli ◽  
Plant Tissue ◽  
Vigna Radiata ◽  
Mung Bean ◽  
Fluorescent Protein ◽  
Vascular Bundles ◽  
Escherichia Coli O157 ◽  
Cortical Cells ◽  
E Coli ◽  
Immunocytochemical Techniques

Escherichia coli O157:H7 has been associated with numerous outbreaks involving fresh produce. Previous studies have shown that bacteria can be internalized within plant tissue and that this can be a source of protection from antimicrobial chemicals and environmental conditions. However, the types of tissue and cellular locations the bacteria occupy in the plant following internalization have not been addressed. In this study, immunocytochemical techniques were used to localize internalized E. coli O157:H7 expressing green fluorescent protein in germinated mung bean (Vigna radiata) hypocotyl tissue following contamination of intact seeds. An average of 13 bacteria per mm3 were localized within the sampled tissue. The bacteria were found to be associated with every major tissue and corresponding cell type (cortex, phloem, xylem, epidermis, and pith). The cortical cells located on the outside of the vascular bundles contained the majority of the internalized bacteria (61%). In addition, the bacteria were localized primarily to the spaces between the cells (apoplast) and not within the cells. Growth experiments were also performed and demonstrated that mung bean plants could support the replication of bacteria to high levels (107 CFU per plant) following seed contamination and that these levels could be sustained over a 12-day period. Therefore, E. coli O157:H7 can be internalized in many different plant tissue types after a brief seed contamination event, and the bacteria are able to grow and persist within the plant.

Increased Transcription of the Phosphate-Specific Transport System of Escherichia coli O157:H7 after Exposure to Sodium Benzoate

2010 ◽  
Vol 73 (5) ◽  
pp. 819-824 ◽  
Author(s):  
FAITH J. CRITZER ◽  
DORIS H. D'SOUZA ◽  
ARNOLD M. SAXTON ◽  
DAVID A. GOLDEN
Keyword(s):  
Escherichia Coli ◽  
Sodium Benzoate ◽  
Efflux Pump ◽  
Transcriptional Response ◽  
Luria Bertani ◽  
Escherichia Coli O157 ◽  
Bacterial Cells ◽  
E Coli ◽  
Specific Transport ◽  
Pst System

Sodium benzoate is a widely used food antimicrobial in drinks and fruit juices. A microarray study was conducted to determine the transcriptional response of Escherichia coli O157:H7 to 0.5% (wt/vol) sodium benzoate. E. coli O157:H7 grown in 150 ml of Luria-Bertani broth was exposed to 0% (control) and 0.5% sodium benzoate. Each treatment was duplicated and sampled at 0 (immediately after exposure), 5, 15, 30, and 60 min. Total RNA was extracted and analyzed with E. coli 2.0 Gene Chips. Significant ontology categories affected by sodium benzoate exposure were determined with JProGO software. The phosphate-specific transport (Pst) system transports inorganic phosphate into bacterial cells, under phosphate-limited conditions. The Pst system was found to be highly upregulated. Increased expression of the Pst system was observed after the short 5 min of exposure to sodium benzoate; pstS, pstA, pstB, and pstC genes were upregulated more than twofold (linear scale) at 5, 15, 30, and 60 min. Increased expression of several other efflux systems, such as AcrAB-TolC, was also observed. The Pst system may act as an efflux pump under these stress-adapted conditions, as well as increase transport of phosphorus to aid in DNA, RNA, ATP, and phospholipid production. Understanding adaptations of Escherichia coli O157:H7 under antimicrobial exposure is essential to better understand and implement methods to inhibit or control its survival in foods.

scholarly journals Biocontrol of Escherichia coli O157 with O157-Specific Bacteriophages

1999 ◽  
Vol 65 (9) ◽  
pp. 3767-3773 ◽  
Author(s):  
Indira T. Kudva ◽  
Srdjan Jelacic ◽  
Phillip I. Tarr ◽  
Philip Youderian ◽  
Carolyn J. Hovde
Keyword(s):  
Escherichia Coli ◽  
Liquid Culture ◽  
Plaque Formation ◽  
Multiplicity Of Infection ◽  
Escherichia Coli O157 ◽  
Bacterial Cells ◽  
Normal Flora ◽  
E Coli ◽  
Complete Lysis ◽  
Fresh Foods

ABSTRACT Escherichia coli O157 antigen-specific bacteriophages were isolated and tested to determine their ability to lyse laboratory cultures of Escherichia coli O157:H7. A total of 53 bovine or ovine fecal samples were enriched for phage, and 5 of these samples were found to contain lytic phages that grow on E. coliO157:H7. Three bacteriophages, designated KH1, KH4, and KH5, were evaluated. At 37 or 4°C, a mixture of these three O157-specific phages lysed all of the E. coli O157 cultures tested and none of the non-O157 E. coli or non-E. colicultures tested. These results required culture aeration and a high multiplicity of infection. Without aeration, complete lysis of the bacterial cells occurred only after 5 days of incubation and only at 4°C. Phage infection and plaque formation were influenced by the nature of the host cell O157 lipopolysaccharide (LPS). Strains that did not express the O157 antigen or expressed a truncated LPS were not susceptible to plaque formation or lysis by phage. In addition, strains that expressed abundant mid-range-molecular-weight LPS did not support plaque formation but were lysed in liquid culture. Virulent O157 antigen-specific phages could play a role in biocontrol of E. coli O157:H7 in animals and fresh foods without compromising the viability of other normal flora or food quality.

Enzyme-Linked Immunomagnetic Electrochemical Detection of Live Escherichia coli O157:H7 in Apple Juice†

2005 ◽  
Vol 68 (1) ◽  
pp. 146-149 ◽  
Author(s):  
ANDREW G. GEHRING ◽  
SHU-I TU
Keyword(s):  
Escherichia Coli ◽  
Electrochemical Detection ◽  
Apple Juice ◽  
Magnetic Beads ◽  
Matrix Effects ◽  
Escherichia Coli O157 ◽  
Bacterial Cells ◽  
E Coli ◽  
Enzyme Substrate ◽  
Assay Time

We describe the application of enzyme-linked immunomagnetic electrochemistry (ELIME) for the rapid detection of Escherichia coli O157:H7 in buffered apple juice. The ELIME technique entails sandwiching bacterial analyte between antibody-coated magnetic beads and an alkaline phosphatase–conjugated antibody. The beads (with or without bound bacteria) were localized onto the surface of magnetized graphite ink electrodes in a multiwell plate format. The enzyme substrate, 1-naphthyl phosphate, was added, and conversion of substrate to an electroactive product was measured using electrochemical detection. With this technique, detection of whole, live E. coli O157:H7 bacterial cells was achieved with a minimum detectable level of ca. 5 × 103 cells per ml in Tris-buffered saline or buffered apple juice in an assay time of ca. 80 min. With adjustment of pH, the ELIME response for the bacteria in either sampling medium was similar, indicating that apple juice components did not contribute to any discernible sample matrix effects.

Survival of Escherichia coli O157:H7 in Ground Beef Jerky Assessed on Two Plating Media

1998 ◽  
Vol 61 (1) ◽  
pp. 11-13 ◽  
Author(s):  
JUDY A. HARRISON ◽  
MARK A. HARRISON ◽  
RUTH ANN ROSE
Keyword(s):  
Escherichia Coli ◽  
Meat Products ◽  
Ground Beef ◽  
Processed Meat ◽  
Escherichia Coli O157 ◽  
Bacterial Cells ◽  
Salmonella Spp ◽  
Suitable Alternative ◽  
E Coli ◽  
Beef Jerky

Recent outbreaks of food-borne illness due to Salmonella spp. in beef jerky and Escherichia coli O157:H7 in venison jerky, coupled with the fact that a variety of preparation methods and drying procedures abound, raise concern over the safety of processed meat products made in the home. The potential of injured bacterial cells to regain the ability to cause illness is a particular threat with pathogens such as E. coli O157:H7, which is believed to have a low infectious dose. This study examined the efficacy of various methods of jerky preparation in reducing populations of E. coli O157:H7 in ground beef jerky and compared the recovery rate of E. coli O157:H7 on two selective plating media, modified sorbitol MacConkey agar (MSMA) and modified eosin methylene blue agar (MEMB). Populations of E. coli O157:H7 in both heated and unheated samples exhibited a greater decline during drying when a nitrite and salt cure mix was added during jerky preparation. When recovery of E. coli O157:H7 on MSMA and MEMB was compared, a trend toward slightly higher recovery rates with MEMB was observed. On the basis of these results, MEMB is a suitable alternative to MSMA for the recovery of E. coli O157:H7 from heated and dried meat samples similar to beef jerky.

Proliferation of Escherichia coli O157:H7 in Soil-Substitute and Hydroponic Microgreen Production Systems

2015 ◽  
Vol 78 (10) ◽  
pp. 1785-1790 ◽  
Author(s):  
ZHENLEI XIAO ◽  
GARY BAUCHAN ◽  
LYDIA NICHOLS-RUSSELL ◽  
YAGUANG LUO ◽  
QIN WANG ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Production System ◽  
Production Systems ◽  
Focal Point ◽  
Peat Moss ◽  
Escherichia Coli O157 ◽  
Bacterial Cells ◽  
E Coli ◽  
Initial Seed ◽  
Whole Plants

Radish (Raphanus sativus var. longipinnatus) microgreens were produced from seeds inoculated with Escherichia coli O157:H7 by using peat moss–based soil-substitute and hydroponic production systems. E. coli populations on the edible and inedible parts of harvested microgreen plants (7 days postseeding) and in growth medium were examined. E. coli O157:H7 was shown to survive and proliferate significantly during microgreen growth in both production systems, with a higher level in the hydroponic production system. At the initial seed inoculation level of 3.7 log CFU/g, E. coli O157:H7 populations on the edible part of microgreen plants reached 2.3 and 2.1 log CFU/g (overhead irrigation and bottom irrigation, respectively) for microgreens from the soil-substitute production system and reached 5.7 log CFU/g for those hydroponically grown. At a higher initial inoculation of 5.6 log CFU/g seeds, the corresponding E. coli O157:H7 populations on the edible parts of microgreens grown in these production systems were 3.4, 3.6, and 5.3 log CFU/g, respectively. Examination of the spatial distribution of bacterial cells on different parts of microgreen plants showed that contaminated seeds led to systematic contamination of whole plants, including both edible and inedible parts, and seed coats remained the focal point of E. coli O157:H7 survival and growth throughout the period of microgreen production.

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