Abstract: E. coli O157 is responsible for a number of human infections through the consumption of foods of animal origin, particularly those originating from cattle. Food poisoning by Escherichia coli O157 causes diarrhea, bloody stools and abdominal pain in humans, and may lead to severe kidney failure or brain injury. For these reasons, E. coli O157 has been the subject of increasingly stringent international regulations. A novel peptide nucleic acid (PNA) array was developed to identify E. coli O157 quickly and accurately. This PNA array exhibited high specificity, significantly detecting E. coli O157 strains without cross-reacting with other bacterial strains. The detection limit of the E. coli O157 PNA array was 100 CFU/ml. The E. coli O157 PNA array is indeed a better option for early detection and accurate diagnosis of the causative agent of a food-borne disease outbreak, which is crucial in planning and implementing strategic measures to prevent and control widespread outbreaks.
Mora A, Usera MA, Blanco M, Blanco JE, Alonso MP, Prats G, Stirrat A, Carter FM, Blanco J (2000). Bacteriophage typing and virulence genes of verocytotoxigenic E. coli (VTEC) O157:H7 strains isolated in Spain. In: Duffy G., Garvey, P., Coia, J., Wasteson, Y., McDowell, D.A. (Eds.), Verocytotoxigenic E. coli in Europe, 3. Pathogenicity and virulence of verocytotoxigenic E. coli. Concerted Action CT98-3935. Teagasc, The National Food Centre, Dublin, pp. 189. Mukherjee A, Speh D, Dyck E, Diez-Gonzalez F (2004). Pre-harvest evaluation of coliforms, Escherichia coli, Salmonella and Escherichia coli O157:H7 in organic and conventional produce grown by Minnesota farmers. J. Food Prot. 67(5): 894–900.
A spatial data-driven stochastic model was developed to explore the spread of verotoxigenic Escherichia coli O157 (VTEC O157) by livestock movements and local transmission among neighbouring holdings in the complete Swedish cattle population. Livestock data were incorporated to model the time-varying contact network between holdings and population demographics. Furthermore, meteorological data with the average temperature at the geographical location of each holding was used to incorporate season. The model was fitted against observed data and extensive numerical experiments were conducted to investigate the model’s response to control strategies aimed at reducing shedding and susceptibility, as well as interventions informed by network measures. The results showed that includ- ing local spread and season improved agreement with prevalence studies. Also, control strategies aimed at reduc- ing the average shedding rate were more efficient in reducing the VTEC O157 prevalence than strategies based on network measures. The methodology presented in this study could provide a basis for developing disease surveillance on regional and national scales, where observed data are combined with readily available high-resolution data in simulations to get an overview of potential disease spread in unobserved regions.
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iTRAQ protein identification and quantification. Raw data files ac- quired from the Orbitrap were converted into MGF files, which were searched against the protein translation database of E. coli O157:H7 EDL933 containing 5,397 sequences by the use of the MASCOT search engine (Matrix Science, London, United Kingdom; version 2.3.02). For protein identification, a mass tolerance of 0.05 Da was permitted for intact peptide masses and a mass tolerance of 0.1 Da for fragmented ions, with allowance for one missed cleavage in the trypsin digests, Gln ¡ pyro-Glu (N-terminal Q), methionine oxidation, and iTRAQ (Y) as potential vari- able modifications and carbamidomethyl modification of cysteine, iTRAQ (N terminus), and iTRAQ (K) as fixed modifications. The charge states of peptides were set to ⫹ 2 and ⫹ 3. Specifically, an automatic decoy database search was performed in Mascot by choosing the decoy checkbox in which a random sequence of database is generated and tested for raw spectra as well as the real database. To reduce the probability of false peptide identification, only peptides with results at the 95% confidence interval that were greater than “identity” as determined by a Mascot prob- ability analysis were counted as identified. Also, each protein identifica- FIG 7 TEM image showing Escherichia coli O157:H7 adhering to HeLa cells.
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Here, we establish zebraﬁsh larvae as a new vertebrate model for EHEC infection. Maintenance of zebraﬁsh is inexpensive, and their propagation and development are quick compared to other vertebrate animals, making them an attractive model organ- ism for infection biology. Zebraﬁsh have been used extensively to study bacterial infections, but few gastrointestinal infection models have been described to date, and our report provides, to our knowledge, the ﬁrst extensive description of a foodborne infection model in zebraﬁsh. Limitations of the zebraﬁsh model are that the microbiota of humans and the microbiota of zebraﬁsh are quite different, although they share some characteristics in that they act as a colonization barrier against pathogens and are stable and yet individually variable. P. caudatum is an ideal vector for foodborne infections: it is commonly used as a food source for young zebraﬁsh, and interactions between E. coli and paramecia have been characterized previously (39). Another limitation of the current model is that the adaptive immune response is not yet fully mature at the larval stage (4 to 10 dpi), and so its contribution to infection remains to be elucidated. In agreement with earlier studies, we found no detrimental effect of EHEC-associated virulence factors on P. caudatum proliferation. E. coli is taken up into food vacuoles within seconds to minutes, depending on the density of suspended particles (40). Vacuoles gradually acidify from an initial pH of 8 to reach a pH of close to 1 (40), and EHEC have a half-life of approximately 150 min under those conditions. The bacterial passage through an acidifying compartment and the subsequent release into the zebraﬁsh foregut upon ingestion of paramecia mimic passage through the human GI tract and acidiﬁcation in the stomach.
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Analysis of genomic DNA of E. coli O157 strains was performed by random amplification of polymorphic DNA (RAPD) PCR fingerprinting with a single primer, primer 1247 (5⬘-AAG AGC CCG T-3⬘) (16). Internal standards (PCR products with known sizes) were run in each lane for standardization of gels for analysis. Gels were stained with ethidium bromide and were digitized for com- puter-aided analysis. The GelCompar software package (Applied Maths, Kor- trijk, Belgium) was used for analysis. Calculation of the similarity matrix was performed with the Jacquard algorithm after defining each single band. The hierarchic clustering was achieved by the unweighted pair group method with arithmetic averages clustering algorithm (36).
(e.g. allicin) are removed from the garlic EO (Hughes and Lawson, 1991). Allicin exhibits its antimicrobial activity primarily through immediate and total inhibition of RNA synthesis; however, DNA and protein syntheses are partially inhibited (Feldberg et al., 1988). Also, the level of lipid content of cell membranes is one of the reasons behind the effect of allicin and other garlic constituents on Gram-negative bacteria (Sivam, 2001). Our findings demonstrated that the MIC and MBC values of the nanoliposome encapsulated garlic EO against E. coli O157:H7 were lower than those of non- encapsulated garlic EO, indicating more susceptibility of the bacteria in nanoliposome encapsulated garlic EO group in comparison with the non-encapsulated one. It is stated that the nanoencapsulation process improves the biological activities of EOs, through development of their bioavailability because of increasing surface to volume ratio by decreasing particle size into nano scale (Ribeiro- Santos et al., 2017). In a similar study, Biddeci et al. (2016) indicated that a bionanocomposite film containing peppermint EO showed enhancing antibacterial effect on E. coli and Staphylococcus aureus, which is in accord- ance with the present work. Also, Beyki et al. (2014) found an improved antifungal property of pepper mint EO encapsulated in chitosan-cinnamic acid nanogels. Similar results have been reported by Makwana et al. (2014) and also Mekkerdchoo et al. (2009).
The catabolism of D-serine by E. coli has been linked to virulence. E. coli catabolise D-serine to use as a nutrient and causes UTI (Alteri & Mobley, 2012, Roesch et al., 2003). D-serine is synthesised by D-serine deaminase or dehydratase, dsdA, to pyruvate and further synthesis produces hydroxypyruvate which is used in the glycolytic pathway (McFall, 1964, Anfora & Welch, 2006, Schell, 2004). Genes encoding the D-serine tolerance locus, dsdCXA, implicates the catabolism of D-serine and triggers virulence (infection). The locus includes a transporter (dsdX) and a dehydratase (dsdA) which is regulated by a LysR-type transcriptional regulator (LTTR) (dsdC) (Nørregaard-Madsen et al., 1995, Anfora & Welch, 2006, Connolly et al., 2016). Also, a D-serine sensory locus yhaOMKJ includes an inner membrane transporter (yhaO) and a LTTR (yhaJ) which modulates the gene expression of virulence factors such as the pathogenicity island LEE (Connolly et al., 2016, Connolly & Roe, 2016). However, the presence of D-serine can inhibit the expression of LEE and yhaJ and in turn affect the colonisation of E. coli in the intestinal tract of the host (Connolly et al., 2015, Fitzhenry et al., 2002, Connolly et al., 2016). The SP E. coli O157 isolates utilised D-serine indicating the expression of dsdCXA and subsequent repression of the yhaJ gene. The SP E. coli O157 isolates may lack the ability to colonise but can survive in the intestinal tract of the host. Furthermore, the preceding PCR were negative for the eae gene (attaching and effacing lesions), which is a part of the LEE (Caprioli et al., 2005), except for isolate EcCa26a which was found to be positive for the gene. However, the SN E. coli isolates did not respire on D-serine indicating the repression of dsdCXA and the expression of the yhaJ gene. The preceding PCR for the SN E. coli O157 isolates were positive for the eae gene. The SN E. coli O157 isolates may possess the ability to colonise the intestinal tract of the host and thus be more virulent than the SP E. coli O157 isolates.
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The proposal of such a dietary switch to reduce E. coli O157:H7 shedding provoked a great deal of scientific controversy (Diez-Gonzalez et al., 1998, Hancock et al., 2000) and led to several studies that have subsequently evaluated the effect of radical dietary shifts on E. coli populations in cattle, however these studies have also produced conflicting results (Table 1). When cattle were fed a high-concentrate diet and switched to a diet containing 50% corn silage and 50% alfalfa hay, generic E. coli counts decreased (Jordan and McEwen, 1998). Cattle fed an 80% barley ration (which as shown previously tends to increase EHEC shedding) were fasted for 48 h and then subsequently switched to 100% alfalfa silage did not exhibit any change in E. coli O157:H7 shedding (Buchko et al., 2000b). However, when these same forage-fed animals were again fasted for 48 h and re-fed 100% alfalfa silage, the prevalence of E. coli O157:H7 shedding increased significantly (Buchko et al., 2000b). In a study using experimentally infected cattle, Researchers found that cattle fed hay shed E. coli O157:H7 significantly longer than did grain-fed cattle (42 d vs. 4 d), but E. coli O157:H7 populations shed were similar between dietary regimes and the diet shift did not affect the acid resistance of E. coli O157:H7 (Hovde et al., 1999). When cattle were abruptly switched from a finishing diet that contained wet corn gluten feed to alfalfa hay for 5 d, colonic pH increased almost 1 pH unit, total E. coli populations decreased approximately 10-fold (Scott et al., 2000). These authors concluded “increased colonic pH was not associated with reduced populations of acid resistant E. coli” but “feeding hay for a short duration can reduce acid- resistant E. coli populations” (Scott et al., 2000).
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pathogenic mechanisms, clinical symptomologies, and virulence factors, pathogenic E. coli strains are classiﬁed into different pathotypes (2, 3). Enterohemorrhagic E. coli (EHEC), in particular, serotype O157:H7, is a unique human pathogen which is associ- ated with severe complications and high mortality compared to other pathotypes. In addition, the primary reservoir for EHEC includes ruminants and the main route of infection involves ingestion of contaminated food products which often result in outbreaks. Infections caused by EHEC can range from acute self-resolving diarrheal episodes to hemorrhagic colitis, which can progress to hemolytic-uremic syndrome (HUS) (2, 3). The production of Shiga toxins (Stx) is one of the deﬁning characteristics of EHEC O157:H7 pathogenesis and the key virulence factor associated with HUS. However, the ﬁrst step in the pathogenic process of EHEC is adherence to intestinal epithelial cells (IECs) (1, 4). To colonize the intestinal mucosa, EHEC O157:H7 is known to utilize a myriad of pili and/or ﬁmbriae to attach to IECs (4). Subsequently, EHEC binding results in an intimate attachment to the surface of IECs and injection of virulence factors by the use of its type III secretion system (TTSS) apparatus. The translocated virulence factors are responsible for the formation of histological changes known as attaching and effacing (A/E) lesions on epithelial cells. These lesions are characterized by the disruption of the microvilli and the accumulation of actin below the site of bacterial adherence to create a “cup-like structure” (2). The genetic factors necessary for the biogenesis of a functional T3SS and of many of its effectors are harbored by a pathogenicity island called the locus of enterocyte effacement (LEE) (5).
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the accumulation of certain toxins associated with these organisms and cause disease con- ditions. Some strains of E. coli particularly produce powerful toxins that can cause intes- tinal or extraintestinal diseases (Kaper and others 2004). These strains include the verotoxin-producing E. coli (VTEC) also known as shiga toxin-producing E. coli (STEC) (Bettelheim and Beutin 2003). The STEC that causes haemorrhagic colitis and haemolytic uraemic syndrome is called enter- ohemorrhagic E. coli (Nataro and Kaper 1998), and it is recognised as an important foodborne pathogen (Gyles 2007). Even healthy animals can harbour human enteric pathogens, many of which have a low infec- tious dose (Bell and others 1994). Although cattle are the main reservoirs of human pathogenic VTEC, there is evidence that sheep, deer, dogs, poultry and goats can also carry the VTEC strains (Callaway and others 2006, Reinstein and others 2007, Greenland and others 2009). E. coli organisms are also classi ﬁ ed into serogroups based on the heat- stable ‘ O ’ and heat-labile surface ‘ K ’ or ﬂ a- gellar ‘ H ’ antigens (Vosti and others 1964). The E. coli O157:H7 serogroup is the most important cause of severe foodborne ill- nesses in living organisms and severe infec- tion can result in case fatality of up to 50 per cent. This serogroup among others carries the shigatoxin 1and 2 and the gene respon- sible for effacement (eae gene) (Gannon and others 1993).
R. officinalis, commonly known as rosemary, is widely known as a spice and used in traditional medicine (20,21). It is a member of the mint family Lamiaceae and is native to Asia and some other parts of the world. Also, C. cyminum can be used in the treatment of digestive and pulmonary diseases (22,23). Considering a wide range of medicinal herbs in Iran, the present study was conducted to examine the effect of the concentrated essential oils of Z. multiflora, R. officinalis and C. cyminum on the growth rate of E.coli O157: H7.
SF E. coli O157 strains without stx genes that carry eae alleles are categorized as atypical EPEC strains and were shown by WGS in this study to be genetically diverse with the exception of SF O157 strains possessing fliCH7 and sfpA encoding Sfp fimbriae, which belong to the O157:H7 CC11. All of the other eae-positive E. coli O157 strains with fliCH16, -H34, -H39, or -H45 are distinct from the O157:H7 complex; these clones are also distinct from each other. This finding is in agreement with the genetic diversity re- ported for other atypical EPEC strains (14, 35). In our previous study, we showed that atypical EPEC strains consist, in general, of two major groups (12, 14). The first group includes strains of a limited spectrum of serotypes that are also frequent among EHEC strains, share non-Stx virulence determinants with such EHEC strains, and cluster into the same MLST CC, demonstrating their close relatedness to EHEC strains. These atypical EPEC serotypes include O26:H11/H ⫺ , O103:H2/H ⫺ , O111:H8/H ⫺ , O121:H19, O145:H28/H ⫺ , and O157:H7/H ⫺ and are presumably EHEC- LST, i.e., EHEC strains that lost their stx genes either during in- fection or during laboratory passages (12, 14). We showed in pre- vious studies that both acquisition and loss of stx-encoding phages is possible and actually happens. Friedrich et al. showed that the loss of stx-encoding phages in O157 is not a rare phenomenon but happens in more than 10% of the cases (13). Later, we demon- strated that even acquisition of stx-encoding phages is possible (36). To do so, we used SF O157 isolates that lost stx from patients and showed that these isolates were able to take up stx-encoding phages. The typical insertion site of such phages in SF O157, yecE,
Unpasteurized fruit juice has been implicated in outbreaks of Escherichia coli O157:H7 (E. coli O157:H7). Meanwhile, certain fruit, such as pomegranate, contains antimicrobial components. The objective of this study was to investigate the survival and growth characteristics of E. coli O157:H7 on pomegranate juice in laboratory medium and in pome- granate-carrot and pomegranate-apple blend juices at different concentrations. Single strain of E. coli O157:H7 (E0019, H1730, and Cider) was inoculated into brain heart infusion (BHI) broth or a mixture of three strains was inoculated into the blended juices containing pomegranate juice. Our results showed that the addition of pomegranate juice inhib- ited the growth of tested E. coli O157:H7 in both laboratory medium and blended juices. The antimicrobial activity in- creased with increased concentrations of pomegranate juice (P < 0.001) and incubation times. The bacterial popula- tion was reduced by at least 2 log CFU/ml in BHI broth and juice blend samples in the presence of 20% and 40% Pome- granate juice respectively. Sensory evaluations performed using a 9 point hedonic scale showed significant sat- isfac- tion on using 40% pomegranate juice blend with carrot and apple juices. Our study suggests that pomegranate juice could be used as a natural antimicrobial in different food systems including juices to inhibit the growth of E. coli O157:H7.
Escherichia coli infections and poor nutritional status have implications on the growth and development of children under five years, physically, mentally and health wise with consequences such as diarrhoea, stunting, wasting, underweight and often times leading to death, depending on their severity. This study evaluated the antibiogram of Escherichia coli O157 and Verocytotoxigenic Escherichia coli (VTEC)and the nutritional status of diarrhoeic children under five years in Kaduna State, Nigeria, using Conventional isolation methods, latex agglutination tests, VTEC-ELISA tests, Chi-square (SPSS Version 19) and WHO Antro (Version 3.2.2). Purposive sampling was used to select 350 children presenting with diarrhoea in six government hospitals within the three senatorial zones of Kaduna State. The results obtained revealed that 76(21.7%) of the 350 stool samples were positive for E. coli and 28(36.8%) were positive for E. coli O157:H7serotype and 1(1.3%) verocytotoxigenic E. coli (VTEC) serotype. High susceptibility to ciprofloxacin, chloramphenicol and high resistance to sulphamethoxazole, cefotaxime, amoxicillin, gentamicin and tetracycline by the
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European Union legislation requires member states to keep national databases of all bovine animals. This allows for disease spread models that includes the time-varying contact network and population demographic. However, per- forming data-driven simulations with a high degree of detail are computationally challenging. We have developed an efficient and flexible discrete-event simulator SimInf for stochastic disease spread modelling that divides work among multiple processors to accelerate the computations. The model integrates disease dynamics as continuous-time Markov chains and livestock data as events. In this study, all Swedish livestock data (births, movements and slaughter) from July 1st 2005 to December 31st 2013 were included in the simulations. Verotoxigenic Escherichia coli O157:H7 (VTEC O157) are capable of causing serious illness in humans. Cattle are considered to be the main reservoir of the bacteria. A better understanding of the epidemiology in the cattle population is necessary to be able to design and deploy targeted measures to reduce the VTEC O157 prevalence and, subsequently, human exposure. To explore the spread of VTEC O157 in the entire Swedish cattle population during the period under study, a within- and between- herd disease spread model was used. Real livestock data was incorporated to model demographics of the population. Cattle were moved between herds according to real movement data. The results showed that the spatial pattern in prevalence may be due to regional differences in livestock movements. However, the movements, births and slaugh- ter of cattle could not explain the temporal pattern of VTEC O157 prevalence in cattle, despite their inherently distinct seasonality.
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Isolation of STEC from bovine reservoirs from other parts of the country has already been documented (8, 21-24). Zahraee Salehi and his colleagues identified STEC O157 among 7 isolates (11.5%), from cattle, whereas non-O157 strains that are frequently associated with sporadic cases of HUS (25, 26), were isolated from 4 (6%) of animals. They showed 5 (8.2%) isolates carried stx genes (21). This finding was in parallel with presence of stx 1 in 35.5 and stx 2 in 49.1% of human isolates (19). This is in contrast with Askari et al. finding with a report of stx 1 and stx 2 , among 5% and 1.9% of calves respectively (23). Recently Ludwig Kerstin et al. reported that 71% of children with HUS were due to Stx2-producing E. coli strains (16). In a study in the USA, stx 1 gene was not detected in any strains tested while 93.1 % of the isolates were found to carry the stx 2 gene (27).
different PFGE patterns, yet, a clear phenotypic and genotypic diversity was noticed among the tested isolates. In the current study, six out of the 10 investigated strains including O157 and non-O157 serotypes shared the same mrp (type I). Sixteen types of antibiotics were tested to examine their resistance profile, and their antibiogram indicated that all 6 STEC strains developed a multi- drug resistance (MDR) pattern to at least four classes of antibiotics including Amoxicillin/Clavulanic Acid 30, Penicillin 10. Spectinomycin Erythromycin 15, Gentamicin 120, Cefazolin 30, Thiampinicol 30, Vancomycin 30, Ciprofloxacin 10. Our study indicated that MDR patterns were more pronounced in STEC isolated from clinical samples such as UTI (O157:EC255,O158:EC158), as well as from meat product (O158:EC306) where resistance to 13 types of antibiotics were noted among these strains, a finding alarm to a serious impact in limiting the selection of treatment drug. This finding corroborated with the study reported in (Mubita et al., 2008), who reported that both clinical and environmental strains displayed MDR phenotype to most of the previously mentioned antibiotics. Many authors documented that the use of antibiotics is strongly associated with the prevalence of antimicrobial resistance in E. coli isolates in food-producing animals (Kang et al., 2005). Similar finding has been reported in other E. coli pathovar in many other studies from Egypt (Putnam et al., 2004, Shaheen et al., 2004) and different parts of the world (Hoge et al.,1998, Okeke et al.,2000, Shapiro et al., 2001, Turner et al., 1988) There is an increasing isolation rate of MDR
Nucleotide sequence analysis of E. coli O114 O-antigen gene cluster. E. coli O114 type strain G1088 (O114:H32) (12) was obtained from the Institute of Medical and Veterinary Science, Adelaide, Australia, and grown under aeration for 12 h in Luria-Bertani broth at 37°C. Chromosomal DNA was pre- pared as described previously (4). Long-range PCR was per- formed with the Expand Long Template PCR system (Roche Applied Science) with primers 1523 (5⬘-ATT GTG GCT GCA GGG ATC AAA GAA AT-3⬘) and 1524 (5⬘-TAG TCG CGT GNG CCT GGA TTA AGT TCG C-3⬘), which were designed based on galF and gnd, respectively (30). PCR was performed as follows: denaturation at 94°C for 10 s, annealing at 60°C for 30 s, and extension at 68°C for 15 min for 30 cycles. The PCR products were digested with DNase I, and the resulting DNA fragments were cloned into pGEM-T Easy (Promega) to pro- * Corresponding author. Mailing address: TEDA School of Biolog-
IMPORTANCE The microbiota protects the host from invading pathogens by limit- ing access to nutrients. In turn, bacterial pathogens selectively exploit metabolites not readily used by the microbiota to establish infection. Ethanolamine has been linked to pathogenesis of diverse pathogens by serving as a noncompetitive meta- bolite that enhances pathogen growth as well as a signal that modulates virulence. Although ethanolamine is abundant in the gastrointestinal tract, the prevailing idea is that commensal bacteria do not utilize EA, and thus, EA utilization has been par- ticularly associated with pathogenesis. Here, we provide evidence that two human commensal Escherichia coli isolates readily utilize ethanolamine to enhance growth, modulate gene expression, and outgrow the pathogen enterohemorrhagic E. coli. These data indicate a more complex role for ethanolamine in host-microbiota- pathogen interactions.