impact of e-coli on food specific (meat), writing homework help

User Generated

fgnezrzr

Writing

Description

My assignment about impact of e-coli on food specific (meat) you should read the 4 articles I attached them in the file you can open it and see them. the last file, I put highlight on some sentences that related with my topic. you will see it when you read.

some notes,

I want you to writer 2 pages. Literature review

Do not write any introduction.

please do not forget to write the references ,

Unformatted Attachment Preview

ORIGINAL RESEARCH ARTICLE published: 06 June 2013 doi: 10.3389/fcimb.2013.00020 CELLULAR AND INFECTION MICROBIOLOGY Phage biocontrol of enteropathogenic and shiga toxin-producing Escherichia coli in meat products David Tomat 1*, Leonel Migliore 1 , Virginia Aquili 1 , Andrea Quiberoni 2 and Claudia Balagué 1 1 2 Área de Bacteriología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina Facultad de Ingeniería Química, Instituto de Lactología Industrial (UNL - CONICET), Santa Fe, Argentina Edited by: Nora L. Padola, Universidad Nacional del Centro de la Provincia de Buenos Aires, Argentina Reviewed by: Mohamed H. Abdulla, Cochin University of Science and Technology, India Adriana Bentancor, Universidad de Buenos Aires, Argentina *Correspondence: David Tomat, Área de Bacteriología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Santa Fe, Argentina e-mail: dtomat@fbioyf.unr.edu.ar Ten bacteriophages were isolated from faeces and their lytic effects assayed on 103 pathogenic and non-pathogenic Enterobacteriaceae. Two phages (DT1 and DT6) were selected based on their host ranges, and their lytic effects on pathogenic E. coli strains inoculated on pieces of beef were determined. We evaluated the reductions of viable cells of Escherichia coli O157:H7 and non-O157 Shiga toxigenic E. coli strains on meat after exposure to DT6 at 5 and 24◦ C for 3, 6, and 24 h and the effect of both phages against an enteropathogenic E. coli strain. Significant viable cell reductions, compared to controls without phages, at both temperatures were observed, with the greatest decrease taking place within the first hours of the assays. Reductions were also influenced by phage concentration, being the highest concentrations, 1.7 × 1010 plaque forming units per milliliter (PFU/mL) for DT1 and 1.4 × 1010 PFU/mL for DT6, the most effective. When enteropathogenic E. coli and Shiga toxigenic E. coli (O157:H7) strains were tested, we obtained viable cell reductions of 0.67 log (p = 0.01) and 0.77 log (p = 0.01) after 3 h incubation and 0.80 log (p = 0.01) and 1.15 log (p = 0.001) after 6 h. In contrast, all nonpathogenic E. coli strains as well as other enterobacteria tested were resistant. In addition, phage cocktail was evaluated on two strains and further reductions were observed. However, E. coli bacteriophage insensitive mutants (BIMs) emerged in meat assays. BIMs isolated from meat along with those isolated by using the secondary culture method were tested to evaluate resistance phenotype stability and reversion. They presented low emergence frequencies (6.5 × 10−7 –1.8 × 10−6 ) and variable stability and reversion. Results indicate that isolated phages were stable on storage, negative for all the virulence factors assayed, presented lytic activity for different E. coli virotypes and could be useful in reducing Shiga toxigenic E. coli and enteropathogenic E. coli viable cells in meat products. Keywords: Escherichia coli, bacteriophage, phage biocontrol, bacteriophage insensitive mutant, phage cocktail INTRODUCTION Shiga toxin-producing Escherichia coli (STEC) are human pathogens that can cause diarrhea, as well as severe clinical manifestations including hemorrhagic enterocolitis, hemolytic uremic syndrome (HUS), and thrombotic thrombocytopenic purpura (Su and Brandt, 1995; Griffin et al., 2002; Yoon and Hovde, 2008). STEC produce several virulence factors which contribute to their pathogenicity. Shiga toxins (Stx), AB type toxins that inhibit protein synthesis in target cells, are the most characterized virulence factors (Thorpe et al., 2002). Shiga toxins produced in the intestines by STEC are able to enter the systemic circulation causing severe damage to distal organs. The degree of damage is related to the amount of toxin produced during the infection (Ritchie et al., 2003). STEC synthesize two main types of Shiga toxins encoded by stx1 and stx2 genes. Moreover, the enterocyte attaching-and-effacing lesion gene (eaeA), which is also present in enteropathogenic strains (EPEC), can contribute to the virulence of STEC. The gene codes for the intimin protein, which allows bacteria to attach themselves to the intestinal epithelium (Frankel et al., 1998). Frontiers in Cellular and Infection Microbiology Foodborne disease-producing Enterobacteriaceae, such as Shigella spp., Salmonella spp., EPEC and STEC, are important etiologic agents of infantile gastroenteritis in Argentina (Binsztein et al., 1999; Rivas et al., 2008). In developing countries, EPEC are the cause of outbreaks of infantile diarrhea with high mortality in children under two years of age. In Argentina, HUS is endemic, with approximately 400 new cases being reported annually by National Health Surveillance System (Rivas et al., 2006), and more than 7000 cases being reported since 1965 (NCASP, 1995). In 2005, the annual incidence of HUS is 13.9 cases/100,000 children under five years of age (Rivas et al., 2006). Recent epidemiological studies showed that there is a sustained global increase in the isolation of non-O157 STEC strains from humans (Tozzi et al., 2003; Brooks et al., 2005; Bettelheim, 2007) and animals (Jenkins et al., 2003; Fernandez et al., 2009), particularly STEC of serogroups O26, O103, and O111 (Ogura et al., 2007). The therapeutic potential of bacteriophages has been explored since they were discovered by Felix d’Herelle (Summers, 1999). Some of the attributes that make bacteriophages interesting as tools for biological control are: (i) their ability to infect and lyse www.frontiersin.org June 2013 | Volume 3 | Article 20 | 1 Tomat et al. Phage biocontrol of Escherichia coli specific bacterial target cells and their inability to infect eukaryotic cells; (ii) phages generally do not cross bacterial species or genus barriers, and therefore do not affect desirable microorganisms commonly present in foods, the gastrointestinal tract or the normal bacterial microbiota (Carlton et al., 2005); (iii) phages need a bacterial host in which to multiply and therefore will persist only as long as the sensitive host is present (Clark and March, 2006). The potential of bacteriophages to control food pathogens is reflected in recent studies involving various pathogens including Campylobacter jejuni (Atterbury et al., 2003; Bigwood et al., 2008), E. coli O157:H7 (O’Flynn et al., 2004; Abuladze et al., 2008) and Listeria monocytogenes (Leverentz et al., 2003; Guenther et al., 2009; Holck and Berg, 2009). Several strategies are currently being applied to preserve perishable refrigerated foods and extend their shelf-life. However, physical processes and chemical compounds (preservatives) used for this purpose may alter meat organoleptic properties. Although bacteriophages represent a novel approach, there are no reports of their industrial use to improve safety, even if this “new, ecological, and specific” technology may be cheaper than “older” technologies, since phages can be isolated from the environment and are self-replicating entities. On the other hand, their inclusion into a meat product can be seen as a less aggressive approach. The aim of this work was to isolate phages with specific lytic capacity for E. coli strains in order to determine phage host range and analyze their potential as biocontrol agents for STEC and EPEC strains in beef products. MATERIALS AND METHODS BACTERIOPHAGE ISOLATION AND PREPARATION OF STOCKS E. coli DH5α was used to isolate bacteriophages from fifty stool samples of patients with diarrhea treated at the Centenary Hospital, Rosario. This strain was grown up to an optical absorbance of 1 (A600 = 1) in 10 mL of Hershey broth (8 g/L Bacto nutrient broth, 5 g/L Bacto peptone, 5 g/L NaCl, and 1 g/L glucose) (Difco, Detroit, MI, USA) supplemented with MgSO4 (5 mM) (Cicarelli, San Lorenzo, Santa Fe, Argentina). A portion of faeces (5 g) was added and the culture was incubated for a further 12 h at 37◦ C. Next, chloroform (0.5 mL, Cicarelli) was added and the preparation was mixed and centrifuged at 15,000× g for 10 min. The supernatant was then filtered through a 0.45 μm pore size (Gamafil S.A., Buenos Aires, Argentina) (Kudva et al., 1999). Bacteriophage isolation and purification were performed by the double-layer plaque technique (Balagué et al., 2006). Briefly, aliquots of filtrates (10 and 100 μL) were mixed with 100 μL of recipient strain culture (A600 = 1), three mL of molten soft agar at 45◦ C (Hershey broth supplemented with 5 mM MgSO4 and 0.7% agar) were added to each suspension and the mixture was poured onto pre-solidified Hershey agar plates and incubated overnight at 37◦ C. To isolate and purify phages, well-defined single plaques on the soft agar were picked and placed in 5 mL of Hershey medium supplemented with 5 mM MgSO4 . Tubes were kept at 4◦ C for 2 h and then inoculated with 100 μL of recipient strain culture (A600 = 1). Inoculated tubes were incubated at 37◦ C with intermittent shaking until complete lysis. Next, chloroform (0.1 mL) was added and cultures were centrifuged at 4000× g for 10 min. Phage stocks were stored Frontiers in Cellular and Infection Microbiology at 4◦ C and enumerated by the double-layer plaque technique (Jamalludeen et al., 2007). These steps were repeated three times. Stability of phage stocks was evaluated after two months of storage at 4◦ C. BACTERIOPHAGE AND BACTERIA CHARACTERIZATION Phage electron micrographs were obtained by the procedure of Bolondi et al. (1995). Phage suspensions were concentrated by centrifugation (1 h, 70,000 × g, 5◦ C) and subsequently stained with phosphotungstic acid (2% w/v) (Biopack, Buenos Aires, Argentina). Electron micrographs were obtained using a JEOL 1200 EX II electron microscope (INTA Castelar, Buenos Aires, Argentina) operating at 85 kV. Phage morphologies and dimensions (head diameter, tail length, and diameter) were recorded. Phages and strains of E. coli were tested for the presence of toxin-encoding genes (stx1, Shiga toxin 1; stx2, Shiga toxin 2; eaeA, attaching-and effacing; LT1, thermolabile toxin and ST1, thermostable toxin) of diarrheogenic E. coli by the polymerase chain reaction (PCR) using primers detailed in Table 1 (Pass et al., 2000). PCR conditions were as follows: initial denaturing step at 95◦ C for 2 min, followed by 25 cycles of 95◦ C for 30 s, annealing at 63◦ C for 30 s and elongation at 72◦ C for 30 s, followed by a final step at 72◦ C for 5 min to achieve complete product elongation. E. coli ATCC43889 (stx2 and eaeA), ATCC43890 (stx1), and ATCC43895 (stx1, stx2, and eaeA, and also harboring the stx2 phage, 933W) were used as positive controls, while enterotoxigenic E. coli ATCC35401 was used for LT1 and ST1 genes. E. coli HB101 and ATCC98222 were utilized as negative controls. Amplified products were resolved by electrophoresis using 3% agarose gels in TBE buffer (89 mM Tris borate, 2 mM EDTA, pH 8.0) (Promega, Madison, WI, USA) at 100 V for 3 h. Gels were stained with ethidium bromide (0.5 μg/mL) (Sigma, St. Louis, MO, USA) and PCR products were visualized under UV light. BACTERIOPHAGE SPECIFICITY The host range of each phage was determined by the double layer agar technique using 44 strains isolated from stool Table 1 | Sequences of primers used in this study. Gene Primera Product size (bp) expected stx1 fp: 5 -ACGTTACAGCGTGTTGCRGGGATC-3 121 bp: 5 -TTGCCACAGACTGCGTCAGTRAGG-3 fp: 5 -TGTGGCTGGGTTCGTTTATACGGC-3 stx2 102 bp: 5 -TCCGTTGTCATGGAAACCGTTGTC-3 eaeA fp: 5 -TGAGCGGCTGGCATGATGCATAC-3 241 bp: 5 -TCGATCCCCATCGTCACCAGAGG-3 fp: 5 -TGGATTCATCATGCACCACAAGG-3 LT1 360 bp: 5 -CCATTTCTCTTTTGCCTGCCATC-3 fp: 5 -TTTCCCCTCTTTTAGTCAGTCAACTG-3 ST1 160 bp: 5 -GGCAGGACTACAACAAAGTTCACAG-3 a fp, forward primer; bp, backward primer. stxl and stx2: Shiga toxin1 and 2 encoding genes; eaeA: intimin encoding gene; LTl and STl: thermolabile and thermostable toxins encoding genes. www.frontiersin.org June 2013 | Volume 3 | Article 20 | 2 Tomat et al. Phage biocontrol of Escherichia coli samples, and urine cultures (uropathogenic E. coli, UPEC). Stool and urine samples were streaked in Cystine Lactose Electrolyte Deficient (CLED) agar plates. Simmons citrate agar test was performed on growing lactose positive colonies. After incubation for 24 h at 35◦ C, only lactose positive and citrate negative colonies were further identified using API system (Biomerieux, Bs. As., Argentina). Sixteen E. coli strain from food (Balagué et al., 2006), one uropathogenic E. coli strain (E. coli T149) which expresses fimbriae P and α-hemolysin (Balagué et al., 2004) and five ATCC E. coli strains were also tested (ATCC 43890; 43889; 43895; 35401 and 98222). Previously characterized (API system) isolates from stool samples were also tested: Shigella flexneri, S. sonnei, Proteus mirabilis, Citrobacter freundii, Klebsiella pneumoniae, Salmonella enteritidis, Salmonella Typhi and Salmonella Typhimurium. Strains tested against stock phages are listed in Table 2. Bacteriophage sensitivity was assayed by placing 10 μL of phage suspension on the solidified soft-agar layer inoculated with 100 μL of each bacterial culture, incubated for 24 h at 37◦ C, and the presence of lysis zones or plaques was examined (Goodridge et al., 2003). MEAT ASSAYS Beef from cow hindquarter purchased from retail was aseptically cut into pieces (1 cm2 of surface and 0.4 cm thick), placed in petri dishes and pre-equilibrated to 5 or 24◦ C. The required pH was obtained by washing with sodium chloride-magnesium sulfate (SM) buffer (0.05 M TRIS, 0.1 M NaCl, 0.008 M MgSO4 , 0.01% w/v gelatin, pH = 7.5) prior to inoculation with bacteria and phage. Host strains employed in this study, namely nonO157 STEC (ARG4827; serogroup O18; harboring stx1 and stx2 genes) (Balagué et al., 2006), O157:H7 STEC (464; harboring stx1 and eaeA genes) and an EPEC (EPEC920; which harbors Table 2 | Strains tested against stock phages. Source Strains (amount) Strains characteristics/ description Food Escherichia coli (10) 8 non-O157 STEC and 2 O157:H7 STEC Stool sample Escherichia coli (9) 4 O157:H7 STEC and 5 EPEC Non-pathogenic Escherichia coli (18) Shigella spp. Salmonella spp. Proteus mirabilis Other enterobacteria Citrobacter freundii Klebsiella pneumoniae (17) Urine culture Escherichia coli (17) UPEC ATCC Escherichia coli (5) 35401; 43889; 43890; 43895 and 98222 eaeA gene), were grown in Hershey medium supplemented with MgSO4 (5 mM) for 12 h at 37◦ C. Bacterial strains and specific bacteriophages added to the meat samples are detailed in Table 3. Twenty μL of each diluted bacterial suspension (ranging from 5.9 × 105 to 3.9 × 107 CFU/mL) were pipetted onto the surface of each meat piece and allowed to attach for 10 min at room temperature. Another 20 μL of each bacteriophage were then pipetted on the meat, at low multiplicity of infection (MOI), 1.7 × 109 PFU/mL for DT1 and 1.4 × 109 PFU/mL for DT6, or high MOI, 1.7 × 1010 PFU/mL for DT1 and 1.4 × 1010 PFU/mL for DT6. Pieces of meat were also added with SM buffer (pH 7.5), instead of phage suspension, as controls. At 3, 6, and 24 h, meat pieces were transferred to a sterile bag, 5 mL SM buffer were added and samples processed for 2 min in a Stomacher (Seward, London, UK). A 1 mL portion of the stomacher fluid was transferred to a sterile tube and cells were pelleted by centrifugation at 3000× g for 10 min. The supernatant was removed and cells were resuspended in 1 mL SM buffer. Finally, a 0.1 mL sample was removed, serially diluted (102 –104 -fold) in SM buffer and 0.1 mL volumes of each dilution were plated on Hershey agar for viable cell enumeration (Bigwood et al., 2008). Phage cocktail (DT1 and DT6 in equal proportions) was assayed on E. coli DH5α (indicator strain used for phage isolation) and in O157:H7 STEC (464) using the methodology employed for each individual phage described above. Three replicates were performed for each treatment and one meat piece processed for replicate. Uninoculated controls were tested to determine the presence of naturally occurring bacteriophages. Plaques (PFU/mL) were evaluated by the double layer agar technique (Jamalludeen et al., 2007). BACTERIOPHAGE INSENSITIVE MUTANTS (BIMs) ISOLATION Bacteriophage insensitive mutants (BIMs) were isolated by the secondary culture method described by Guglielmotti et al. (2007) with some modifications. E. coli sensitive strains (one EPEC, three O157:H7 STEC and one non-O157 STEC) (A600 = 0.2 − 0.3) were infected with a phage suspension at different infection ratios (multiplicity of infection, MOI of ≈ 10 and 1), incubated in Hershey broth at 37◦ C for 24 h and observed visually until complete lysis. An uninfected culture of each E. coli strain was used as a control. Cultures exhibiting complete and delayed lysis were the best candidates to isolate BIMs. After lysis, further incubation for 48 h at 37◦ C was required for secondary growth. Each tube with secondary growth was spread on Hershey agar plates for colony isolation. BIMs were isolated from meat as described in meat assays methodology described above modified with an extended incubation time (48 h) at 37◦ C. For both of the aforementioned methodologies, after incubation of agar plates, eight different colonies were randomly isolated (on agar plates) and cultured overnight in Hershey broth at 37◦ C. These isolates were purified by three consecutive streakings on Hershey agar plates. The growing colonies were isolated as presumptive BIMs. EPEC, enteropathogenic E. coli; O157:H7 STEC, O157:H7 Shigatoxigenic E. coli; BIMs CONFIRMATION non-O157 STEC, Shigatoxigenic non-O157 E. coli; UPEC, urophatogenic E. coli; Presumptive BIMs were confirmed by a liquid culture sensitivity test (Guglielmotti et al., 2007). Briefly, a log-phase culture ATCC, american type culture collection. Frontiers in Cellular and Infection Microbiology www.frontiersin.org June 2013 | Volume 3 | Article 20 | 3 Tomat et al. Phage biocontrol of Escherichia coli Table 3 | E. coli viable cell logarithmic reductions after phage treatment of contaminated meat products. Phage stock/sensitive strain DT1/EPEC (920) DT6/EPEC (920) DT6/non-O157 STEC (ARG4827) DT6/O157:H7 STEC (464) Cocktail/DH5α Cocktail/O157:H7 STEC (464) Log reduction in E. coli viable cellsa after the incubation time (h)b Assay conditions T (◦ C) MOI 3 6 24 5 4.4 × 102 NS **0.80 ± 0.14 NS 24 4.8 × 102 **0.30 ± 0.05 NS NS 5 4.4 × 101 NS **0.49 ± 0.09 NS 24 4.8 × 101 NS NS **0.46 ± 0.08 5 5.2 × 102 **0.67 ± 0.12 **0.59 ± 0.11 *0.46 ± 0.15 24 6.5 × 103 *0.32 ± 0.09 NS NS 5 5.2 × 101 NS *0.30 ± 0.08 NS 24 6.5 × 102 NS NS NS 5 2.4 × 104 *0.33 ± 0.09 *0.47 ± 0.12 *0.56 ± 0.17 24 4.0 × 102 *0.43 ± 0.13 NS NS 5 2.4 × 103 NS *0.37 ± 0.09 *0.50 ± 0.16 24 4.0 × 101 *0.35 ± 0.11 NS NS 5 2.3 × 103 *0.59 ± 0.16 **0.86 ± 0.15 *0.38 ± 0.10 24 5.8 × 103 **0.77 ± 0.14 ***1.15 ± 0.12 NS 5 2.3 × 102 *0.38 ± 0.09 *0.62 ± 0.18 NS 24 5.8 × 102 NS **0.74 ± 0.13 NS 5 2.25 × 104 *0.91 ± 0.19 **2.16 ± 0.20 **2.23 ± 0.21 24 1.75 × 104 *0.66 ± 0.15 NS NS 5 1.56 × 105 NS NS NS 24 3.33 × 105 **1.43 ± 0.24 **2.58 ± 0.21 **2.20 ± 0.22 MOI, multiplicity of infection (PFU/CFU); NS, not significant. Mean values of treated and control samples not significantly different using the scheffé method (*significant at p = 0.05; **significant at p = 0.01; ***significant at p = 0.001). a Log reduction in E. coli viable cells with respect to phage-free control. b Mean of three data points ± standard deviations. (A600 = 0.2 − 0.3) of each presumptive BIM in Hershey broth was infected with the phage suspension at various MOI (≈ 10 and 1). Uninfected cultures of each E. coli strain were used as controls. BIMs cultures were incubated in Hershey broth at 37◦ C until growth of control strains was evident. Infected cultures that did not lyse at the first attempt were subcultured again. Each second subculture was prepared by transferring 2–3% of the final volume from the first culture to another test tube with 1 mL of fresh broth. When no bacterial lysis was evident, the resulting culture was stored at 4◦ C and subcultured under the same conditions. Presumptive BIMs that survived the third subculture were considered to be confirmed BIMs. Sensitivity of each parent strain (sensitive) was always determined in parallel to ensure lytic activity of phage suspensions. mixture was supplemented with MgSO4 (5 mM), plated by the double-layer agar technique and incubated overnight at 37◦ C. BIM frequency was estimated as the ratio of the number of confirmed BIM to the initial bacterial number. All the experiments were performed in duplicate. Selected BIMs were propagated through 50 generations at 37◦ C and then checked by a plaque assay to evaluate reversion to phage sensitivity (O’Flynn et al., 2004). Phage resistance stability was assayed by seven sequential subcultures of 2% portions of BIM cultures (Hershey broth) with independent addition of phage at each subculture (Guglielmotti et al., 2007). The loss of phage resistance was determined by comparing lysis of BIM culture with the control (mutant subculture without phage addition). The subculture where lysis first occurred was recorded. DETERMINATION OF BACTERIOPHAGE-INSENSITIVE MUTANT FREQUENCY, REVERSION, AND STABILITY STATISTICAL ANALYSIS The emergence frequency of BIMs was evaluated by mixing the appropriate volume of an overnight culture of each strain (EPEC920 and O157:H7 STEC 464) and phage suspension (DT1 and DT6) to obtain a MOI of 100. The bacterium–phage Frontiers in Cellular and Infection Microbiology Means and standard deviations for data sets were calculated. Differences between means for control (untreated) and treated samples were compared by the Scheffé method and Origin 6.0 for graphics. Differences were considered statistically significant when p-values were
Purchase answer to see full attachment
User generated content is uploaded by users for the purposes of learning and should be used following Studypool's honor code & terms of service.

Explanation & Answer

Attached.

Running head: LITERATURE REVIEW ON IMPACT OF E-COLI ON FOOD (MEAT)

Literature Review on Impact of E-Coli on Food (Meat)
Name
Institution

1

LITERATURE REVIEW ON IMPACT OF E-COLI ON FOOD (MEAT)

2

Literature Review on Impact of E-Coli on Food (Meat)
In 1982, E. coli O157:H7 was for the first time recognized to be a food borne pathogen.
The consumption of meat contaminated with the pathogen has been associated with a number of
health problems. For example, E. coli O157:H7 was associated with an outbreak of hemorrhagic
colitis, a condition associated with bloody diarrhea in the United States of America in 1982 (E.
coli bacteria, 2016). Medical investigators found that the outbreak was as a result of the use of
meat contaminated with E. coli O157:H7 in hamburgers. The E. coli O157:H7 bacteria have also
been known to cause Hemolytic Uremic Syndrome (HUS). This occurs after the infection has
remained untreated for a long duration of time. Research has shown that HUS oft...


Anonymous
Great content here. Definitely a returning customer.

Studypool
4.7
Trustpilot
4.5
Sitejabber
4.4

Similar Content

Related Tags