Escherichia coli O157:H7 Cells Exposed to Lettuce Leaf Lysate in Refrigerated Conditions Exhibit Differential Expression of Selected Virulence and Adhesion-Related Genes with Altered Mammalian Cell Adherence

2016 ◽  
Vol 79 (7) ◽  
pp. 1259-1265 ◽  
Author(s):  
NICOLE M. KENNEDY ◽  
NABANITA MUKHERJEE ◽  
PRATIK BANERJEE
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Mammalian Cells ◽  
Pathogenic Bacteria ◽  
Cold Shock ◽  
Cold Shock Protein ◽  
Toxin Gene ◽  
Adhesion Assay ◽  
Escherichia Coli O157 ◽  
The Impact

ABSTRACT Contamination by and persistence of pathogenic bacteria in ready-to-eat produce have emerged as significant food safety and public health concerns. Viable produceborne pathogens cope with several stresses (e.g., temperature fluctuations and low-temperature storage) during production and storage of the commodities. In this study, we investigated the impact of transient cold shock on Escherichia coli O157:H7 (EcO157) cells in a produce matrix (romaine lettuce leaf lysate). EcO157 cells were exposed to 25°C for 1 h, 4°C for 1 h, and 4°C for 10 min in lettuce lysate. The expression of selected genes coding for virulence, stress response, and heat and cold shock proteins was quantified by real-time quantitative reverse transcription PCR assay. Treated EcO157 cells adhered to MAC-T mammalian cells were enumerated by in vitro bioassay. Expression of the Shiga toxin 1 gene (stx1a) was upregulated significantly (P < 0.05) upon cold shock treatments, but virulence genes related to EcO157 attachment (eaeA, lpfA, and hcpA) were down-regulated. Two key members of the cold shock regulon, cold shock protein (cspA) and gyrA, were significantly induced (P < 0.05) at the refrigeration temperature (4°C). Significant upregulation of an SOS response gene, recA, was also observed. E. coli heat shock regulon member grpE was induced, but a universal stress protein (uspA) was down-regulated at the refrigeration temperatures in lettuce lysate. The adhesion assay revealed a temperature-dependent reduction in the attachment of cold-shocked EcO157 cells. The results of the current study indicate a reduction in the attachment of cold-shocked EcO157 to epithelial cells and higher levels of Shiga toxin gene expression at the molecular level.

scholarly journals Comparison between human and animal isolates of Shiga toxin-producing Escherichia coli O157 from Australia

2002 ◽  
Vol 128 (3) ◽  
pp. 357-362 ◽  
Author(s):  
N. FEGAN ◽  
P. DESMARCHELIER
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Human Infection ◽  
Pulsed Field Gel Electrophoresis ◽  
Toxin Gene ◽  
Escherichia Coli O157 ◽  
Animal Population ◽  
E Coli ◽  
Virulence Markers ◽  
Animal Isolates

There is very little human disease associated with enterohaemorrhagic Escherichia coli O157 in Australia even though these organisms are present in the animal population. A group of Australian isolates of E. coli O157:H7 and O157:H- from human and animal sources were tested for the presence of virulence markers and compared by XbaI DNA macrorestriction analysis using pulsed-field gel electrophoresis (PFGE). Each of 102 isolates tested contained the gene eae which encodes the E. coli attaching and effacing factor and all but one carried the enterohaemolysin gene, ehxA, found on the EHEC plasmid. The most common Shiga toxin gene carried was stx2c, either alone (16%) or in combination with stx1 (74%) or stx2 (3%). PFGE grouped the isolates based on H serotype and some clusters were source specific. Australian E. coli O157:H7 and H- isolates from human, animal and meat sources carry all the virulence markers associated with EHEC disease in humans therefore other factors must be responsible for the low rates of human infection in Australia.

scholarly journals Molecular analysis of Escherichia coli O157 strains isolated from cattle and pigs by the use of PCR and pulsed-field gel electrophoresis methods

2012 ◽  
Vol 47 (No. 6) ◽  
pp. 149-158 ◽  
Author(s):  
J. Osek ◽  
P. Gallien
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Genetic Relatedness ◽  
Rflp Analysis ◽  
Toxin Gene ◽  
Escherichia Coli O157 ◽  
Chromosomal Dna ◽  
E Coli ◽  
Shiga Toxin Genes ◽  
Porcine Origin

Fourteen Escherichia coli O157 strains isolated from cattle and pigs in Poland and in Germany were investigated, using PCR, for the genetic markers associated with Shiga toxin-producing E. coli (STEC). Only two strains, both of cattle origin, were positive for the fliC (H7) gene and could be classified as O157 : H7. Nine isolates had stx shiga toxin genes, either stx1 (1 strain), stx2 (4 isolates) or both (4 strains). The stx2-carrying samples were further subtyped by PCR for the stx2c, stx2d, and stx2e toxin variants. It was shown that all but one stx2-positive bacteria possessed the stx2c Shiga toxin gene type and one stx2 STEC isolate had the stx2d virulence factor sub-type. The eaeA (intimin) gene was found in 9 strains (8 isolates from cattle and one strain from pigs); all of them harboured the genetic marker characteristic of the gamma intimin variant. The translocated intimin receptor (tir) gene was detected in 7 isolates tested and among them only one tir-positive strain was recovered from pigs. The ehly E. coli enterohemolysin gene was amplified in all but one strains obtained from cattle and only in one isolate of porcine origin. The genetic relatedness of the analysed E. coli O157 strains was examined by restriction fragment length polymorphism (RFLP) of chromosomal DNA digested with XbaI. Two distinct but related RFLP pattern clusters were observed: one with 9 strains (8 isolates of bovine origin and one strain obtained from pigs) and the other one comprises the remaining 5 E. coli isolates (4 of porcine origin and one strain recovered from cattle). The results suggest that pigs, besides cattle, may be a reservoir of E. coli O157 strains potentially pathogenic to humans. Moreover, epidemiologically unrelated isolates of the O157 serogroup, recovered from different animal species, showed a clonal relationship as demonstrated by the RFLP analysis.

Efficacy of Chemical Treatments in Eliminating Salmonella and Escherichia coli O157:H7 on Scarified and Polished Alfalfa Seeds

2001 ◽  
Vol 64 (10) ◽  
pp. 1489-1495 ◽  
Author(s):  
SARAH L. HOLLIDAY ◽  
ALAN J. SCOUTEN ◽  
LARRY R. BEUCHAT
Keyword(s):  
Escherichia Coli ◽  
Chemical Treatment ◽  
Organic Material ◽  
Pathogenic Bacteria ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Mechanical Abrasion ◽  
Chemical Treatments ◽  
The Impact ◽  
Alfalfa Seeds

Alfalfa seeds are sometimes subjected to a scarification treatment to enhance water uptake, which results in more rapid and uniform germination during sprout production. It has been hypothesized that this mechanical abrasion treatment diminishes the efficacy of chemical treatments used to kill or remove pathogenic bacteria from seeds. A study was done to compare the effectiveness of chlorine (20,000 ppm), H2O2 (8%), Ca(OH)2 (1%), Ca(OH)2 (1%) plus Tween 80 (1%), and Ca(OH)2 (1%) plus Span 20 (1%) treatments in killing Salmonella and Escherichia coli O157:H7 inoculated onto control, scarified, and polished alfalfa seeds obtained from two suppliers. The influence of the presence of organic material in the inoculum carrier on the efficacy of sanitizers was investigated. Overall, treatment with 1% Ca(OH)2 was the most effective in reducing populations of the pathogens. Reduction in populations of pathogens on seeds obtained from supplier 1 indicate that chemical treatments are less efficacious in eliminating the pathogens on scarified seeds compared to control seeds. However, the effectiveness of chemical treatment in removing Salmonella and E. coli O157:H7 from seeds obtained from supplier 2 was not markedly affected by scarification or polishing. The presence of organic material in the inoculum carrier did not have a marked influence on the efficacy of chemicals in reducing populations of test pathogens. Additional lots of control, scarified, and polished alfalfa seeds of additional varieties need to be tested before conclusions can be drawn concerning the impact of mechanical abrasion on the efficacy of chemical treatment in removing or killing Salmonella and E. coli O157:H7.

scholarly journals Reactivation of Insertionally Inactivated Shiga Toxin 2 Genes of Escherichia coli O157:H7 Caused by Nonreplicative Transposition of the Insertion Sequence

2000 ◽  
Vol 66 (3) ◽  
pp. 1133-1138 ◽  
Author(s):  
Masahiro Kusumoto ◽  
Yoshiaki Nishiya ◽  
Yoshihisa Kawamura
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Insertion Sequence ◽  
Pcr Amplification ◽  
Direct Repeat ◽  
Toxin Gene ◽  
Escherichia Coli O157 ◽  
Wild Type ◽  
In The Wild ◽  
Shiga Toxin 2

ABSTRACT IS1203v is an insertion sequence which has been found in inactivated Shiga toxin 2 genes of Escherichia coliO157:H7. We analyzed the transpositional mechanism of IS1203v in order to investigate whether the Shiga toxin 2 genes inactivated by IS1203v could revert to the wild type. When the transposase activity of IS1203v was enhanced by artificial frameshifting, IS1203v was obviously excised from the Shiga toxin 2 gene in a circular form. The IS1203v circle consisted of the entire IS1203v, but an extra 3-bp sequence (ATC) intervened between the 5′ and 3′ ends of IS1203v. The extra 3-bp sequence was identical to a direct repeat which was probably generated upon insertion. Moreover, we detected the Shiga toxin 2 gene with a precise excision of IS1203v. In the wild-type situation, the transposition products of IS1203v could be observed by PCR amplification. These results show that IS1203v can transpose in a nonreplicative manner and that the Shiga toxin gene inactivated by this insertion sequence can revert to the wild type.

scholarly journals Electroceutical disinfection strategies impair the motility of pathogenic Pseudomonas aeruginosa and Escherichia coli

2016 ◽  
Author(s):  
Kristina Doxsee ◽  
Ryan Berthelot ◽  
Suresh Neethirajan
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Acetic Acid ◽  
Mammalian Cells ◽  
Pathogenic Bacteria ◽  
Electrical Potential ◽  
Microfluidic Platform ◽  
E Coli ◽  
Therapeutic Benefits ◽  
The Impact

Electrotaxis or galvanotaxis refers to the migration pattern of cells induced in response to electrical potential. Although it has been extensively studied in mammalian cells, electrotaxis has not been explored in detail in bacterial cells; information regarding the impact of current on pathogenic bacteria is severely lacking. Therefore, we designed a series of single and multi-cue experiments to assess the impact of varying currents on bacterial motility dynamics in pathogenic multi-drug resistant (MDR) strains of Pseudomonas aeruginosa and Escherichia coli using a microfluidic platform. Motility plays key roles in bacterial migration and the colonization of surfaces during the formation of biofilms, which are inherently recalcitrant to removal and resistant to traditional disinfection strategies (e.g. antibiotics). Use of the microfluidic platform allows for exposure to current, which can be supplied at a range that is biocidal to bacteria, yet physiologically safe in humans (single cue). This system also allows for multi-cue experiments where acetic acid, a relatively safe compound with anti-fouling/antimicrobial properties, can be combined with current to enhance disinfection. These strategies may offer substantial therapeutic benefits, specifically for the treatment of biofilm infections, such as those found in the wound environment. Furthermore, microfluidic systems have been successfully used to model the unique microfluidic dynamics present in the wound environment, suggesting that these investigations could be extended to more complex biological systems. Our results showed that the application of current in combination with acetic acid has profound inhibitory effects on MDR strains of P. aeruginosa and E. coli, even with brief applications. Specifically, E. coli motility dynamics and cell survival were significantly impaired starting at a concentration of 125 μA DC and 0.31% acetic acid, while P. aeruginosa was impaired at 70 μA and 0.31% acetic acid. As these strains are relevant wound pathogens, it is likely that this strategy would be effective against similar strains in vivo and could represent a new approach to hasten wound healing.

A report on the prevalence and the whole-genome sequence-based analysis of Shiga-toxin producing Escherichia coli isolated from the recto-anal junction of slaughter-age Irish sheep

2021 ◽  
Author(s):  
Siobhán C. McCarthy ◽  
Guerrino Macori ◽  
Gina Duggan ◽  
Catherine M. Burgess ◽  
Séamus Fanning ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Human Disease ◽  
Pathogenic Bacteria ◽  
Small Ruminants ◽  
Whole Genome Sequence ◽  
Health Concern ◽  
Toxin Gene ◽  
Whole Genome ◽  
Faecal Excretion

Shiga toxin-producing Escherichia coli (STEC) are a diverse group of pathogenic bacteria capable of causing serious human illness and serogroups O157 and O26 are frequently implicated in human disease. Ruminant hosts are the primary STEC reservoir and small ruminants are important contributors to STEC transmission. This study investigated the prevalence, serotypes and shedding dynamics of STEC, including the super-shedding of serogroups O157 and O26, in Irish sheep. Recto-anal mucosal swab samples (N=840) were collected over 24 months from two ovine slaughtering facilities. Samples were plated on selective agars and were quantitatively and qualitatively assessed via real-time PCR for Shiga-toxin prevalence and serogroup. A subset of STEC isolates (N=199) were selected for whole-genome sequencing and analysed in silico . In total, 704/840 (83.8%) swab samples were Shiga-toxin positive following RT-PCR screening, and 363/704 (51.6%) animals were subsequently culture positive for STEC. Five animals were shedding STEC O157 and three of these were identified as super-shedders. No STEC O26 was isolated. Post-hoc statistical analysis showed that younger animals are more likely to harbour STEC and STEC carriage is most prevalent during the summer months. Following sequencing, 178/199 genomes were confirmed as STEC. Thirty-five different serotypes were identified, fifteen of which were not yet reported in sheep. Serotype O91:H14 was the most frequently reported. Eight Shiga-toxin gene variants were reported, two stx 1 and six stx 2 , and three novel Shiga-toxin subunit combinations were observed. Variant stx 1c was the most prevalent, while many strains also harboured stx 2b . Importance Shiga toxin-producing Escherichia coli (STEC) are foodborne, zoonotic pathogens of significant public health concern. All STEC harbour stx , a critical virulence determinant, but it is not expressed in most serotypes. Sheep shed the pathogen via faecal excretion and are increasingly recognised as important contributors in the dissemination of STEC. In this study, we have found that there is high prevalence of STEC circulating within sheep and prevalence is related to animal age and seasonality. Further, sheep harbour a variety of non-O157 STEC, whose prevalence and contribution to human disease has been under investigated for many years. A variety of Stx variants were also observed, some of which are of high clinical importance.

scholarly journals Escherichia coli O157:H7 Strain Origin, Lineage, and Shiga Toxin 2 Expression Affect Colonization of Cattle

2009 ◽  
Vol 75 (15) ◽  
pp. 5074-5081 ◽  
Author(s):  
Ross M. S. Lowe ◽  
Danica Baines ◽  
L. Brent Selinger ◽  
James E. Thomas ◽  
Tim A. McAllister ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Escherichia Coli O157 ◽  
Human Origin ◽  
E Coli ◽  
Unknown Factor ◽  
Shiga Toxin 2 ◽  
The Impact ◽  
Dose Dependent

ABSTRACT Enterohemorrhagic Escherichia coli O157:H7 has evolved into an important human pathogen with cattle as the main reservoir. The recent discovery of E. coli O157:H7-induced pathologies in challenged cattle has suggested that previously discounted bacterial virulence factors may contribute to the colonization of cattle. The objective of the present study was to examine the impact of lineage type, cytotoxin activity, and cytotoxin expression on the amount of E. coli O157:H7 colonization of cattle tissue and cells in vitro. Using selected bovine- and human-origin strains, we determined that lineage type predicted the amount of E. coli O157:H7 strain colonization: lineage I > intermediate lineages > lineage II. All E. coli O157:H7 strain colonization was dose dependent, with threshold colonization at 103 to 105 CFU and maximum colonization at 107 CFU. We also determined that an as-yet-unknown factor of strain origin was the most dominant predictor of the amount of strain colonization in vitro. The amount of E. coli O157:H7 colonization was also influenced by strain cytotoxin activity and the inclusion of cytotoxins from lineage I or intermediate lineage strains increased colonization of a lineage II strain. There was a higher level of expression of the Shiga toxin 1 gene (stx 1) in human-origin strains than in bovine-origin strains. In addition, lineage I strains expressed higher levels of the Shiga toxin 2 gene (stx 2). The present study supports a role for strain origin, lineage type, cytotoxin activity, and stx 2 expression in modulating the amount of E. coli O157:H7 colonization of cattle.

scholarly journals Survival of Shiga toxin-producing Escherichia coli O157 in marine water and frequent detection of the Shiga toxin gene in marine water samples from an estuary port

2001 ◽  
Vol 126 (1) ◽  
pp. 129-133 ◽  
Author(s):  
K. MIYAGI ◽  
K. OMURA ◽  
A. OGAWA ◽  
M. HANAFUSA ◽  
Y. NAKANO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Water Samples ◽  
Marine Water ◽  
Toxin Gene ◽  
Escherichia Coli O157

Shiga toxin-producing Escherichia coli (STEC) O157 was investigated with respect to its halotolerance and whether it can survive in marine water. STEC O157 could multiply in a medium containing 5% NaCl and in sterilized marine water, and could survive in unsterilized marine water for at least 15 days. On the basis of these results, we postulated that STEC O157 may survive in natural marine water, and attempted to isolate the bacterium and Shiga toxin gene (stx) from marine water in Japan. The stx, comprising stx1 and stx2, was detected from marine water samples by PCR. STEC and other stx-positive bacteria, however, could not be isolated from these samples in this study. These results indicate that stx-positive bacteria may survive in marine water and suggest the necessity of a survey.

Association of Prophage Antiterminator Q Alleles and Susceptibility to Food-Processing Treatments Applied to Escherichia coli O157 in Laboratory Media

2007 ◽  
Vol 70 (11) ◽  
pp. 2617-2619 ◽  
Author(s):  
AARON S. MALONE ◽  
AHMED E. YOUSEF ◽  
JEFFREY T. LEJEUNE
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Food Processing ◽  
High Pressure Processing ◽  
Toxin Gene ◽  
Escherichia Coli O157 ◽  
Surrogate Markers ◽  
Processing Efficiency ◽  
E Coli ◽  
Q Gene

Resistance of Escherichia coli O157 to inactivation by high-pressure processing, heat, and UV and gamma radiation was associated with the allele of the prophage-encoded antiterminator Q gene present upstream of the Shiga toxin gene stx2. Increased processing may be required to kill certain strains of E. coli O157, and the choice of strains used as surrogate markers for processing efficiency is critical.

scholarly journals Global effects of homocysteine on transcription in Escherichia coli: induction of the gene for the major cold-shock protein, CspA

Microbiology ◽  
2006 ◽  
Vol 152 (8) ◽  
pp. 2221-2231 ◽  
Author(s):  
Katy R. Fraser ◽  
Nina L. Tuite ◽  
Arvind Bhagwat ◽  
Conor P. O'Byrne
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Growth Medium ◽  
Amino Acid Metabolism ◽  
Cold Shock ◽  
Acid Metabolism ◽  
Cold Shock Protein ◽  
Rt Pcr ◽  
Life Threatening ◽  
The Impact

Homocysteine (Hcy) is a thiol-containing amino acid that is considered to be medically important because it is linked to the development of several life-threatening diseases in humans, including cardiovascular disease and stroke. It inhibits the growth of Escherichia coli when supplied in the growth medium. Growth inhibition is believed to arise as a result of partial starvation for isoleucine, which occurs because Hcy perturbs the biosynthesis of this amino acid. This study attempted to further elucidate the inhibitory mode of action of Hcy by examining the impact of exogenously supplied Hcy on the transcriptome. Using gene macroarrays the transcript levels corresponding to 68 genes were found to be reproducibly altered in the presence of 0.5 mM Hcy. Of these genes, the biggest functional groups affected were those involved in translation (25 genes) and in amino acid metabolism (19 genes). Genes involved in protection against oxidative stress were repressed in Hcy-treated cells and this correlated with a decrease in catalase activity. The gene showing the strongest induction by Hcy was cspA, which encodes the major cold-shock protein CspA. RT-PCR and reporter fusion experiments confirmed that cspA was induced by Hcy. Induction of cspA by Hcy was not caused by nutritional upshift, a stimulus known to induce CspA expression, nor was it dependent on the presence of a functional CspA protein. The induction of cspA by Hcy was suppressed when isoleucine was included in the growth medium. These data suggest that the induction of CspA expression in the presence of Hcy occurs because of a limitation for isoleucine. The possibility that Hcy-induced cspA expression is triggered by translational stalling that occurs when the cells are limited for isoleucine is discussed.

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