Evaluation by Flow Cytometry of Escherichia coli Viability in Lettuce after Disinfection

Foodborne outbreaks due to the consumption of ready-to-eat vegetables have increased worldwide, with Escherichia coli (E. coli) being one of the main sources responsible. Viable but nonculturable bacteria (VBNC) retain virulence even after some disinfection procedures and constitute a huge problem to public health due to their non-detectability through conventional microbiological techniques. Flow cytometry (FCM) is a promising tool in food microbiology as it enables the distinction of the different physiological states of bacteria after disinfection procedures within a short time. In this study, samples of lettuce inoculated with E. coli were subject to disinfection with sodium hypochlorite at free chlorine concentrations of 5, 10, 25, 50, and 100 mg·L−1 or with 35% peracetic acid at concentrations of 5, 10, 25, and 50 mg·L−1. The efficiency of these disinfectants on the viability of E. coli in lettuce was evaluated by flow cytometry with LIVE/DEAD stains. Results from this study suggest that FCM can effectively monitor cell viability. However, peracetic acid is more effective than sodium hypochlorite as, at half the concentration, it is enough to kill 100% of bacteria and always induces a lower percentage of VBNC. Finally, we can conclude that the recommended levels of chemical disinfectants for fresh fruit and vegetables are adequate when applied in lettuce. More importantly, it is possible to ensure that all cells of E. coli are dead and that there are no VBNC cells even with lower concentrations of those chemicals. These results can serve as guidance for lettuce disinfection, improving quality and the safety of consumption.

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PCR for the Specific Detection of an Escherichia coli O157:H7 Laboratory Control Strain

Journal of Food Protection ◽

10.4315/0362-028x.jfp-15-147 ◽

2015 ◽

Vol 78 (9) ◽

pp. 1738-1744 ◽

Cited By ~ 6

Author(s):

MICHAEL KNOWLES ◽

DOMINIC LAMBERT ◽

GEORGE HUSZCZYNSKI ◽

MARTINE GAUTHIER ◽

BURTON W. BLAIS

Keyword(s):

Escherichia Coli ◽

Pathogenic Bacteria ◽

Control Measure ◽

Positive Control ◽

Food Microbiology ◽

Escherichia Coli O157 ◽

Control Strain ◽

E Coli ◽

Signature Sequences ◽

Materials Used

Control strains of bacterial pathogens such as Escherichia coli O157:H7 are commonly processed in parallel with test samples in food microbiology laboratories as a quality control measure to assure the satisfactory performance of materials used in the analytical procedure. Before positive findings can be reported for risk management purposes, analysts must have a means of verifying that pathogenic bacteria (e.g., E. coli O157:H7) recovered from test samples are not due to inadvertent contamination with the control strain routinely handled in the laboratory environment. Here, we report on the application of an in-house bioinformatic pipeline for the identification of unique genomic signature sequences in the development of specific oligonucleotide primers enabling the identification of a common positive control strain, E. coli O157:H7 (ATCC 35150), using a simple PCR procedure.

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Molecular Characterization of Plasmid-Mediated Non-O157 Verotoxigenic Escherichia coli Isolated from Infants and Children with Diarrhea

Baghdad Science Journal ◽

10.21123/bsj.2020.17.3.0710 ◽

2020 ◽

Vol 17 (3) ◽

pp. 0710

Author(s):

Md Fazlul Karim Khan ◽

Shah Samiur Rashid

Keyword(s):

Escherichia Coli ◽

Virulence Gene ◽

Multidrug Resistant ◽

Plasmid Curing ◽

Health Issues ◽

E Coli ◽

Disk Diffusion Assay ◽

Microbiological Techniques ◽

Polymerase Chain

A significant increase in the incidence of non-O157 verotoxigenic Escherichia coli (VTEC) infections have become a serious health issues, and this situation is worsening due to the dissemination of plasmid mediated multidrug-resistant microorganisms worldwide. This study aims to investigate the presence of plasmid-mediated verotoxin gene in non-O157 E. coli. Standard microbiological techniques identified a total of 137 E. coli isolates. The plasmid was detected by Perfectprep Plasmid Mini preparation kit. These isolates were subjected to disk diffusion assay, and plasmid curing with ethidium bromide treatment. The plasmid containing isolates were subjected to a polymerase chain reaction (PCR) for investigating the presence of plasmid mediated verotoxin gene (VT1 and VT2) in non-O157 E. coli. Among the 137 E. coli isolates, 49 isolates were non-O157 E. coli while 29 (59.1%) isolates were verotoxin producing non-O157 serotypes and 26 non-O157 VTEC isolates possessed plasmids. Certain isolates harboured single sized plasmid while others had multiple plasmids with different size varied from 1.8kb to 7.6kb. A plasmid containing all (100%) the isolates was multidrug-resistant. Eight isolates changed their susceptibility patterns while three isolates were found to lose plasmid after post plasmid curing treatment and the rest of the isolates (15) remained constant. Different PCR sets characterized 3 plasmid-mediated verotoxins producing non-O157 E. coli. This current study demonstrated the occurrence of plasmid mediated verotoxin gene in non-O157 E. coli. To the best of our knowledge, this is the first report in the global literature on plasmid-mediated verotoxin gene in non-O157 E. coli. Timely diagnosis and surveillance of VTEC infections should prioritize to stop or slow down the virulence gene for dissemination by plasmid-mediated gene transfer amongst the same bacteria or other species.

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Impact of UV and Peracetic Acid Disinfection on the Prevalence of Virulence and Antimicrobial Resistance Genes in Uropathogenic Escherichia coli in Wastewater Effluents

Applied and Environmental Microbiology ◽

10.1128/aem.00418-14 ◽

2014 ◽

Vol 80 (12) ◽

pp. 3656-3666 ◽

Cited By ~ 24

Author(s):

Basanta Kumar Biswal ◽

Ramzi Khairallah ◽

Kareem Bibi ◽

Alberto Mazza ◽

Ronald Gehr ◽

...

Keyword(s):

Escherichia Coli ◽

Antimicrobial Resistance ◽

Resistance Genes ◽

Peracetic Acid ◽

Content Type ◽

E Coli ◽

Antimicrobial Resistance Genes ◽

Antimicrobial Resistance Gene ◽

Municipal Wastewaters ◽

The Impact

ABSTRACTWastewater discharges may increase the populations of pathogens, includingEscherichia coli, and of antimicrobial-resistant strains in receiving waters. This study investigated the impact of UV and peracetic acid (PAA) disinfection on the prevalence of virulence and antimicrobial resistance genes in uropathogenicEscherichia coli(UPEC), the most abundantE. colipathotype in municipal wastewaters. Laboratory disinfection experiments were conducted on wastewater treated by physicochemical, activated sludge, or biofiltration processes; 1,766E. coliisolates were obtained for the evaluation. The target disinfection level was 200 CFU/100 ml, resulting in UV and PAA doses of 7 to 30 mJ/cm2and 0.9 to 2.0 mg/liter, respectively. The proportions of UPECs were reduced in all samples after disinfection, with an average reduction by UV of 55% (range, 22% to 80%) and by PAA of 52% (range, 11% to 100%). Analysis of urovirulence genes revealed that the decline in the UPEC populations was not associated with any particular virulence factor. A positive association was found between the occurrence of urovirulence and antimicrobial resistance genes (ARGs). However, the changes in the prevalence of ARGs in potential UPECs were different following disinfection, i.e., UV appears to have had no effect, while PAA significantly reduced the ARG levels. Thus, this study showed that both UV and PAA disinfections reduced the proportion of UPECs and that PAA disinfection also reduced the proportion of antimicrobial resistance gene-carrying UPEC pathotypes in municipal wastewaters.

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Impact of Transportation of Feedlot Cattle to the Harvest Facility on the Prevalence of Escherichia coli O157:H7, Salmonella, and Total Aerobic Microorganisms on Hides

Journal of Food Protection ◽

10.4315/0362-028x-70.1.17 ◽

2007 ◽

Vol 70 (1) ◽

pp. 17-21 ◽

Cited By ~ 20

Author(s):

A. L. REICKS ◽

M. M. BRASHEARS ◽

K. D. ADAMS ◽

J. C. BROOKS ◽

J. R. BLANTON ◽

...

Keyword(s):

Escherichia Coli ◽

Feedlot Cattle ◽

Lower Percentage ◽

Escherichia Coli O157 ◽

Sample Collection ◽

Treatment Groups ◽

E Coli ◽

Sampling Locations ◽

Before And After

Prevalences of Escherichia coli O157:H7, Salmonella, and total aerobic microorganisms were determined on the hides of beef feedlot cattle before and after transport from the feedyard to the harvest facility in clean and dirty trailers. Swab samples were taken from the midline and withers of 40 animals on each of 8 days before and after shipping. After samples were collected, animals were loaded in groups of 10 on upper and lower levels of clean and dirty trailers. Animals were unloaded at the harvest facility and kept in treatment groups for sample collection after exsanguination. Salmonella was found more often on hide swabs collected from the midline than on than samples collected from the withers from animals transported in both clean and dirty trailers. Salmonella was found on significantly more hide swabs collected at harvest from both sampling locations than on those collected at the feedyard, with no differences attributed to the type of trailer. At the feedyard, clean trucks had a lower percentage of Salmonella-positive samples than did dirty trucks before animals were loaded. However, after transport, both clean and dirty trucks had a similar prevalence of Salmonella. There were no differences in Salmonella prevalence on hides collected from animals transported on the top and bottom levels of clean and dirty trucks. E. coli O157:H7 was detected on less than 2% of the samples; therefore, no practical conclusions about prevalence could be drawn. Hides sampled at harvest had higher concentrations of aerobic microorganisms than did hides sampled at the feedyard, and concentrations were higher on the midline than on the withers. Although the prevalences of Salmonella and total aerobic microorganisms increased on hides after transport from the feedyard to the plant, this increase was not related to the cleanliness of the trailers or the location of the cattle in the trailers.

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Inactivation of E. coli, Enterococcus spp., somatic coliphage, and Cryptosporidium parvum in wastewater by peracetic acid (PAA), sodium hypochlorite, and combined PAA-ultraviolet disinfection

Environmental Science Water Research & Technology ◽

10.1039/c9ew00837c ◽

2020 ◽

Vol 6 (1) ◽

pp. 197-209 ◽

Cited By ~ 3

Author(s):

Abdulrahman H. Hassaballah ◽

Tanmay Bhatt ◽

Jeremy Nyitrai ◽

Ning Dai ◽

Lauren Sassoubre

Keyword(s):

Sodium Hypochlorite ◽

Cryptosporidium Parvum ◽

Peracetic Acid ◽

Ecosystem Health ◽

Ultraviolet Disinfection ◽

E Coli ◽

Enterococcus Spp ◽

Wastewater Disinfection ◽

Somatic Coliphage

Wastewater disinfection is important to protect human and ecosystem health.

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Inactivation of Multi-Drug Resistant Non-Typhoidal Salmonella and Wild-Type Escherichia coli STEC Using Organic Acids: A Potential Alternative to the Food Industry

Pathogens ◽

10.3390/pathogens9100849 ◽

2020 ◽

Vol 9 (10) ◽

pp. 849

Author(s):

Vinicius Silva Castro ◽

Yhan da Silva Mutz ◽

Denes Kaic Alves Rosario ◽

Adelino Cunha-Neto ◽

Eduardo Eustáquio de Souza Figueiredo ◽

...

Keyword(s):

Escherichia Coli ◽

Acetic Acid ◽

Organic Acids ◽

Sodium Hypochlorite ◽

Bacterial Species ◽

Disk Diffusion ◽

Clinical Strains ◽

Linear Pattern ◽

E Coli ◽

Log Reduction

Salmonella and Escherichia coli are the main bacterial species involved in food outbreaks worldwide. Recent reports showed that chemical sanitizers commonly used to control these pathogens could induce antibiotic resistance. Therefore, this study aimed to describe the efficiency of chemical sanitizers and organic acids when inactivating wild and clinical strains of Salmonella and E. coli, targeting a 4-log reduction. To achieve this goal, three methods were applied. (i) Disk-diffusion challenge for organic acids. (ii) Determination of MIC for two acids (acetic and lactic), as well as two sanitizers (quaternary compound and sodium hypochlorite). (iii) The development of inactivation models from the previously defined concentrations. In disk-diffusion, the results indicated that wild strains have higher resistance potential when compared to clinical strains. Regarding the models, quaternary ammonium and lactic acid showed a linear pattern of inactivation, while sodium hypochlorite had a linear pattern with tail dispersion, and acetic acid has Weibull dispersion to E. coli. The concentration to 4-log reduction differed from Salmonella and E. coli in acetic acid and sodium hypochlorite. The use of organic acids is an alternative method for antimicrobial control. Our study indicates the levels of organic acids and sanitizers to be used in the inactivation of emerging foodborne pathogens.

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Development and Characterization of a Fluorescent-Bacteriophage Assay for Detection of Escherichia coli O157:H7

Applied and Environmental Microbiology ◽

10.1128/aem.65.4.1397-1404.1999 ◽

1999 ◽

Vol 65 (4) ◽

pp. 1397-1404 ◽

Cited By ~ 96

Author(s):

Lawrence Goodridge ◽

Jinru Chen ◽

Mansel Griffiths

Keyword(s):

Escherichia Coli ◽

Flow Cytometry ◽

Immunomagnetic Separation ◽

Escherichia Coli O157 ◽

E Coli ◽

Lower Detection ◽

Unreliable Method ◽

Direct Epifluorescent Filter Technique ◽

Bacterial Concentrations

ABSTRACT In this paper we describe evaluation and characterization of a novel assay that combines immunomagnetic separation and a fluorescently stained bacteriophage for detection of Escherichia coliO157:H7 in broth. When it was combined with flow cytometry, the fluorescent-bacteriophage assay (FBA) was capable of detecting 104 cells/ml. A modified direct epifluorescent-filter technique (DEFT) was employed in an attempt to estimate bacterial concentrations. Using regression analysis, we calculated that the lower detection limit was between 102 and 103cells/ml; however, the modified DEFT was found to be an unreliable method for determining bacterial concentrations. The results of this study show that the FBA, when combined with flow cytometry, is a sensitive technique for presumptive detection of E. coliO157:H7 in broth cultures.

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Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes

The Journal of Lipid Research ◽

10.1194/jlr.ra120000654 ◽

2020 ◽

Vol 61 (6) ◽

pp. 870-883 ◽

Cited By ~ 2

Author(s):

Inga Nilsson ◽

Sheng Y. Lee ◽

William S. Sawyer ◽

Christopher M. Baxter Rath ◽

Guillaume Lapointe ◽

...

Keyword(s):

Escherichia Coli ◽

Gram Negative Bacteria ◽

Metabolic Labeling ◽

Wild Type ◽

Transport Pathways ◽

Gram Negative ◽

E Coli ◽

Outer Membranes ◽

Outer Leaflet ◽

Promising Tool

Gram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPSs) on the outer leaflet and phospholipids (PLs) on the inner leaflet. The loss of this asymmetry due to mutations in the LPS biosynthesis or transport pathways causes the externalization of PLs to the outer leaflet of the OM and leads to OM permeability defects. Here, we used metabolic labeling to detect a compromised OM in intact bacteria. Phosphatidylcholine synthase expression in Escherichia coli allowed for the incorporation of exogenous propargylcholine into phosphatidyl(propargyl)choline and exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC), as confirmed by LC/MS analyses. A fluorescent copper-free click reagent poorly labeled AEPC in intact wild-type cells but readily labeled AEPC from lysed cells. Fluorescence microscopy and flow cytometry analyses confirmed the absence of significant AEPC labeling from intact wild-type E. coli strains and revealed significant AEPC labeling in an E. coli LPS transport mutant (lptD4213) and an LPS biosynthesis mutant (E. coli lpxC101). Our results suggest that metabolic PL labeling with AECho is a promising tool for detecting a compromised bacterial OM, revealing aberrant PL externalization, and identifying or characterizing novel cell-active inhibitors of LPS biosynthesis or transport.

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Inactivation of Stressed Escherichia coli O157:H7 Cells on the Surfaces of Rocket Salad Leaves by Chlorine and Peroxyacetic Acid

Journal of Food Protection ◽

10.4315/0362-028x.jfp-13-019 ◽

2014 ◽

Vol 77 (1) ◽

pp. 32-39 ◽

Cited By ~ 11

Author(s):

ANAS A. AL-NABULSI ◽

TAREQ M. OSAILI ◽

HEBA M. OBAIDAT ◽

REYAD R. SHAKER ◽

SADDAM S. AWAISHEH ◽

...

Keyword(s):

Escherichia Coli ◽

Deionized Water ◽

Escherichia Coli O157 ◽

Apparent Difference ◽

Peroxyacetic Acid ◽

Surface Disinfection ◽

E Coli ◽

Leafy Greens ◽

Foodborne Outbreaks ◽

Stressed Cells

Because Escherichia coli O157:H7 has been frequently associated with many foodborne outbreaks caused by consumption of leafy greens (lettuce, spinach, and celery), this study investigated the ability of deionized water, chlorine, and peroxyacetic acid to detach or inactivate stressed and unstressed cells of E. coli O157:H7 contaminating the surfaces of rocket salad leaves. E. coli O157:H7 cells stressed by acid, cold, starvation, or NaCl exposure, as well as unstressed cells, were inoculated on the surfaces of rocket salad leaves at 4°C. The effectiveness of two sanitizers (200 ppm of chlorine and 80 ppm of peroxyacetic acid) and deionized water for decontaminating the leaves treated with stressed and unstressed E. coli O157:H7 were evaluated during storage at 10 or 25°C for 0.5, 1, 3, and 7 days. It was found that washing with 80 ppm of peroxyacetic acid was more effective and reduced unstressed and stressed cells of E. coli O157:H7 by about 1 log CFU per leaf on the leaves. There was no apparent difference in the ability of stressed and unstressed cells to survive surface disinfection with the tested agents. Treatments to reduce viable E. coli O157:H7 cells on rocket leaves stored at 25°C were more effective than when used on those stored at 10°C. Washing with peroxyacetic acid or chlorine solution did not ensure the safety of rocket leaves, but such treatments could reduce the likelihood of water-mediated transfer of E. coli O157:H7 during washing and subsequent processing.

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Expression of Different-Size Transcripts from theclpP-clpX Operon of Escherichia coli during Carbon Deprivation

Journal of Bacteriology ◽

10.1128/jb.182.23.6630-6637.2000 ◽

2000 ◽

Vol 182 (23) ◽

pp. 6630-6637 ◽

Cited By ~ 13

Author(s):

Chin Li ◽

Yi Ping Tao ◽

Lee D. Simon

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Type Strain ◽

Wild Type Strain ◽

Transcription Termination ◽

Carbon Starvation ◽

Lower Percentage ◽

Wild Type ◽

Lower Efficiency ◽

E Coli

ABSTRACT Transcription of the clpP-clpX operon ofEscherichia coli leads to the production of two different sizes of transcripts. In log phase, the level of the longer transcript is higher than the level of the shorter transcript. Soon after the onset of carbon starvation, the level of the shorter transcript increases significantly, and the level of the longer transcript decreases. The longer transcript consists of the entireclpP-clpX operon, whereas the shorter transcript contains the entire clpP gene but none of the clpXcoding sequence. The RpoH protein is required for the increase in the level of the shorter transcript during carbon starvation. Primer extension experiments suggest that there is increased usage of the ς32-dependent promoter of the clpP-clpXoperon within 15 min after the start of carbon starvation. Expression of the clpP-clpX operon from the promoters upstream of theclpP gene decreases to a very low level by 20 min after the onset of carbon starvation. Various pieces of evidence suggest, though they do not conclusively prove, that production of the shorter transcript may involve premature termination of the longer transcript. The half-life of the shorter transcript is much less than that of the longer transcript during carbon starvation. E. coli rpoBmutations that affect transcription termination efficiency alter the ratio of the shorter clpP-clpX transcript to the longer transcript. The E. coli rpoB3595 mutant, with an RNA polymerase that terminates transcription with lower efficiency than the wild type, accumulates a lower percentage of the shorter transcript during carbon starvation than does the isogenic wild-type strain. In contrast, the rpoB8 mutant, with an RNA polymerase that terminates transcription with higher efficiency than the wild type, produces a higher percentage of the shorter clpP-clpXtranscript when E. coli is in log phase. These and other data are consistent with the hypothesis that the shorter transcript results from premature transcription termination during production of the longer transcript.

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