Biotechnology Analytical Techniques

1 Biotechnology Analytical Techniques Routinely Used Methods for Detect Escherichia coli (E. coli) in Food and Environment Introduction E. coli is a gram negative bacillus enterobacteriaceae that exists as a normal flora in the intestines. Escherichia coli were first identified as a human food pathogen following the 1982 hemorrhagic colitis epidemics. The bacteria were isolated from patients and undercooked hamburgers, and were subsequently linked to the severe gastrointestinal infections, and ever since, intensive research work has been ongoing about E. coli. Currently, more than 200 food borne infections are known across the world, but Escherichia coli remains one of the major pathogens widely studied. The pathogenic strains of E. coli are classified into five groups including, “enterotoxegenic (ETEC), Enteropathogenic (EPEC), Enteroinvasive (EIEC), Enteroaggregative (EAEC), and enterohemorrhagic (EHEC)”

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2 Routine Methods for Detecting E. coli in Food and Environment Pathogenic E. coli cannot be detected using a single method because of the wide phenotypic nature. Therefore, different methods must be used for specific isolation. The most coomon methods used in the detection are culture, immunological-based techniques and polymerase chain reaction (Kaper, Nataro & Mobley 2004).

Culturing-based methods Conventional culture methods are still the most reliable and accurate techniques for detection of E. coli. Culture based techniques are regarded as the standard microbiological methods but require culturing of a single cell into a colony (Lazcka, Campo & Munoz 2007).

The sample for testing must be pre-enriched in a broth at 35°C for three hours, after which it is transferred into a tryptone phosphate (TP) broth and incubated for about 18-20 hours at 44°C. An aliquot from TP broth is then inoculated onto agar mediums like eosin-methylene blue and MacConkey and incubated at room temperature for 24 hours (Efsa 2007). Culture methods often are not sensitive because sometimes the bacteria can be dormant in the environment, thus becoming non-culturable, resulting into poor isolation or underestimation. In addition, the culturing methods are labor demanding and take a lot of time as compared to other methods (Donnenberg 2002).

Immunological methods for detection of E. coli This technique is used to detect E. coli cells and toxins based on antigen-antibody reactions. Immunoassay techniques target the detection of O and H antigens on the cell surface of E. coli (Beutin et al. 1984). Antibodies are coated on Immunomagnetic beads then employed in capturing and isolating the E. coli suspension in a sample. Immunological methods like

3 ELISA are preferable because they optimize on the specificity of antibodies and sensitivity of enzymes (Perelle et al. 2006).

Aim of testing E. coli in environmental and food sources People have become more concerned about the adverse effects resulting from contamination of food by E. coli. In the developed countries, more than 30 percent of the people suffer from food borne illnesses annually, but the problem is more pronounced in the low and middle income countries where poverty renders people more vulnerable to ill health. The current trends in the manufacture, processing, distribution, and preparation of food have posed new challenges in ensuring that food is not harmful for human consumption (Adams & Moss 2008).

Nowadays, foods grown or processed in one part of the world easily reaches other parts of the world. Besides, many food varieties are available today than in the past. The increased life expectancy and high number of individuals tremendously having low immune status increases the number of people at risk of being affected by unsafe foods (Karmali 2003). Safe food enhances the health of the population and is a fundamental right. It promotes productivity and offers a platform for elimination of poverty.

E. coli mostly originate from fecal matter, thus transmission is through contamination of food and water by stool. Transmission can also arise from cross contamination or direct human contact when handling food. Food borne route is usually through consumption of undercooked beef or beef products contaminated with feces. Dairy products like milk and cheese are potential causes of infection due to unhygienic preparation, unpasteurization, or failure of the pasteurization process. Water also may be contaminated with fecal material as a result of poor design or improper treatment, thus making it unsafe (Karmali 2003).

E. coli strain specifically tested

4 The strain specifically tested for strain is O157:H7, the verocytotoxin/Shiga toxinproducing. It is the most common pathogenic E. coli and cause of hemolytic uremic syndrome globally, and only a small infectious dose is required to cause illnesses. The infections from this strain of E. coli are very severe, such as bloody diarrhea and the hemolytic uremic syndrome. All people can be infected by E. coli O157:H7, but the elderly and the children are more vulnerable (Karmali 2003). Though not frequent, hemolytic uremic syndrome is a serious complication that may impair with the functioning of the kidney. Most people infected will develop anemic symptoms and the patients often must be hospitalized and undergo dialysis until the kidney heals. However, in majority of the patients, the kidney functions become permanently or partially impaired after healing.

Detection of E. coli O157:H7 As healthy people normally do not carry enterohemorrhagic E. coli, patients presenting with clinical symptoms are diagnosed by elucidating the bacteria in fecal specimens. To identify the reservoir of the pathogens, analysis of food and environmental samples is also carried out.

The gold standard method for testing Shiga toxin-producing E. coli is culture on Vero cells or other similar ones like HeLa cells. Other methods include immunological testing for the Shiga toxin and molecular methods, which indirectly examine for the genes that encode the toxin vtx (Nataro & Kaper 1998).

Use of culture-based methods Cell cultures are used to examine the presence of free Vero cytotoxin in stool specimens, thus implying infection. They are also employed in enrichment mediums inoculated with foods, animal feces, water and other environmental specimens. Vero cytotoxins cause permanent lysis of the susceptible cells in culture. In carrying out the test, cell-free supernatants are inoculated on

5 a monolayer of culture cells and incubated, then observed for growth. Preliminary results are achieved in 24 hours, while the final examination is done after 3 to 4 days (Nataro & Kaper 1998).

Selective and differential media for enterohemorrhagic E. coli O157:H7 are formulated based on properties such as the slow rate of fermentation of sorbitol by most strains and absence of beta-glucuronidase activity. Various agars like MacConkey, Hemorrhagic colitis agar, and chromogenic agar, are used to for isolation. To differentiate from other microorganisms and other strains of E. coli, prior enrichment is necessary especially in food and environmental specimens (Nataro & Kaper 1998). The enrichment requires culturing in liquid enrichment medium. Immunomagnetic techniques can also be employed in concentrating the O157 strains.

This is achieved by the use of magnetic beads pre-coated with O157 antigen-specific antibodies.

Suspected colonies of the enterohemorrhagic E. coli O157:H7 require confirmation using biochemical tests, after which immunoassays are applied to demonstrate the presence of the flagellar and somatic antigens (Karmali 2003).

The culture method is very sensitive and, thus, is regarded as the gold standard test method. However, the method is very involving and specialized in nature. Besides, the maintenance cell culture is expensive. This has resulted into the adoption of immunological methods that identify the Vero cytotoxins.

Immunological methods for detection of E. coli O157:H7 Immunological test are important in the identification of Vero cytotoxin antigens from different forms of samples and confirmation of the toxin production obtained from pure culture specimens. Immunological approaches employ Vero cytotoxin specific poly- or monoclonal

6 antibodies. The formats available include, “enzyme-linked immunosorbent assays and reversed passive latex agglutination” (Karmali 2003 p. 6).

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Essay 2: Analytical Methods for the Detection and Diagnosis of Infectious Diseases Enzyme linked Immuno Sorbant Assay (ELISA) Enzyme linked immuno sorbant assay (ELISA) is an immunological method used in the identification of antigens and antibodies. ELISA technique can be both qualitative and quantitative; hence it is carried out to demonstrate the presence of specific proteins and their quantity (eBioscience 2012). ELISA exists in two variants depending on whether it is the antibody in a sample which is being determined or it is the protein bound by an antibody. The variation, therefore, allows for quantification of antibodies or other proteins. This technique is highly sensitive and safe since it does not use radioisotopes. Given it does not require use of sophisticated equipments like isotope counters, this technique can be used in small laboratories and can be suited in field environments (Karmali 2003).

The ELISA assay consists of a solid phase that is coated with specific antibodies or antigens, antibody or antigen enzyme conjugate and enzyme substrates. Polystyrene or polyvinyl chloride microtitre plates serve as solid supports. The most commonly used solid supports are nitro cellulose paper and cellulose acetate membrane. The various enzyme conjugates that are used in this assay include, “peroxidase, B-galactosidase, alkaline phosphatase, penicillinase, urease, and glucose oxidase” (Wilson & Walker 2010 p. 216).

ELISAs are carried out usually in 96-well plates in order to enable detailed output.

Different types of assay principles are applicable in ELISA techniques. The major principles are the competitive, sandwich and antibody capture principles. In competitive principles, one component of the immune reaction is immobilized, while the other one is labeled with an enzyme (Crowther 1995). The amount of the analyte is then measured using the analyte’s ability

8 to inhibit the formation of a complex between the immobilized and labeled reagent. This method is advantageous because it requires only a single incubation step. Even at high antibody concentrations, agglutination occurs, thus preventing prozone effect (Wilson & Walker 2010).

This method is, however, limited by the narrow range of concentration to which the analyte sample can be measured without sample dilution. Besides, in instances where either the antigen or antibody is available in the sample, same response is elicited; hence the single step assay cannot give a distinction (eBioscience 2012).

In sandwich ELISA, one component of the immune reaction is used both in immobilized and enzyme labeled states. The other component in the analyte acts as a bridge between the two reagents. Therefore, in this method, one component, for instance the antigen is immobilized and binds the analyte from the sample, usually the antibody. The analyte is then detected through addition of a secondary labeled antibody against the same class of antibody as the primary antibody (Crowther 1995). Sandwich assays allow measurements to be performed on a diverse range of analyte concentration. To prevent prozone effect, the technique requires sequential incubation steps to be done on the sample and label, besides use of monoclonal antibodies (Sadasivam & Manickam 1996). Antibody capture ELISA technique targets the detection of antibodies of particular immunoglobulin subclasses. This is achieved by first allowing the sample to react with the immobilized specific antigen, followed by either enzyme labeled antigen, then enzyme linked antibody.

The ELISA assays are highly sensitive, specific and practical. Sensitivity has been facilitated by the availability of monoclonal and polyclonal antibodies having an extremely high affinity. The assay can also be modified to increase specificity by decreasing the height and

9 variability of the background reactions, hence making very low concentrations of the analyte to be easily quantified (Crowther 2000).

Polymerase Chain Reaction (PCR) Polymerase Chain Reaction (PCR) technique was invented in 1983 by Kary Mullis and is used to amplify a given DNA segment into many copies containing a particular sequence based on the ability of DNA polymerase to produce new strand of DNA complementary to the template strand (Persing 1993). The novelty of this technique is derived from its relative ease in performing it, utilization of simple apparatus like test tubes, simple reagents, and source of heat.

The technique is widely applied in medical and biological fields to perform many functions such as, “DNA cloning for sequencing, DNA-based phylogeny, diagnosis of hereditary diseases, and identification of genetic finger prints in forensic science, and in the diagnosis and detection of infectious diseases” (Latchman 1995 p. 107).

Principle of Polymerase Chain Reaction Polymerase chain reaction technique is founded on the principle of DNA polymerization reaction. It involves repeated thermal cycling of the reaction to enable breakage of bonds in the DNA molecule and enzymatic replication by the use of DNA polymerase, primer sequence and deoxyribonucleotide triphosphates (dNTPs). This allows for amplification of a specific sequence of DNA into many kilo base pairs. The synthesis of DNA starts at the 5’ end, proceeding to the 3’ end, with each primer extending to complete the sequence. The newly formed strand acts as a template for another cycle, thus exponentially amplifying the product (Pelt-Verkuil, Belkum & Hays 2008).

Procedure of Polymerase Chain Reaction

10 The Polymerase chain reaction is mostly designed to perform between 20-40 cycles, each cycle characterized by temperature changes. The cycling is initiated at a hold step of high temperature, followed by another hold in the final product extension. The steps, therefore, involved include the initialization, denaturation, annealing and extension (Pelt-Verkuil, Belkum & Hays 2008). The starting step of the PCR is the initialization step whereby the temperature is increased to an average of 95°C for about 1-9 minutes. This step helps in activating the polymerase to be used in the reaction. In the denaturation step, temperature needed for reaction is maintained at 94-98°C for about half a minute to allow for the breakage of hydrogen bonds, thus separating the double stranded DNA molecules into single stranded (Erlich 1994).

In the annealing phase, the temperature is lowered to between 50-65°C for about half a minute to enable the primers anneal into the ssDNA template. It is necessary that the primer sequence closely resembles the template sequence to facilitate stable annealing. The extension step introduces the enzymes and, therefore, the temperature for the reaction is determined by the DNA polymerase used. The temperature must be readjusted to allow the polymerase form complementary strands using the 3’-OH of the primer. Optimum temperature allows the DNA polymerase to exponentially amplify the specific DNA fragments.

Polymerase chain reaction is a novel molecular technology that comes as an alternative to the traditional methods such as agar based culture techniques, which are time consuming and have relatively low specificity and sensitivity. The technique can be optimized to perform many functions. As compared to culture methods, PCR has more specificity, is fast and low cost, hence its increasing application in different areas. The time required to perform the polymerase chain reaction assay can be in hours, as opposed to culture methods which take several days or weeks.

Polymerase chain reaction can amplify to detectable levels nucleic acids of disease causing

11 organisms from various samples (Molecular Methods in Microbiology 2012). The assay can enable detection of pathogens from large volumes after concentration by filter-adsorption and elution methods.

12 Reference List Adams, MR, & Moss, MO 2008, Food microbiology, 3rd edition, RSC Publishing, Cambridge.

Beutin, L, Bode L, Richter T, Peltre G & Stephan, R 1984, ‘Rapid visual detection of Escherichia coli and vibrio cholera Heat-labile enterotoxins by nitrocellulose enzyme linked immunosorbent assay’, J Clin Microbiol, vol. 19, no. 3, pp. 371-375.

Crowther JR 2000, The ELISA guidebook, Humana Press, Totowa, NJ. Crowther, JR 1995, Elisa: theory and practice, Humana Press, Totowa, NJ Inc. Donnenberg, M 2002, Escherichia coli: Virulence mechanisms of a versatile pathogen, Academic Press, San Diego, CA. eBioscience 2012, Enzyme Linked Immunosorbance Essay (ELISA), viewed December 1, 2012, http://media.ebioscience.com/data/pdf/best-protocols/enzyme-linked-immunosorbent-assayelisa.pdf Efsa 2007, ‘Monitoring of verotoxigenic Escherichia coli (VTEC) and identification of human pathogenic VTEC types’, The EFSA Journal, vol. 579, pp. 1-61.

Erlich, HA 1994, PCR technology: principles and applications for DNA amplification, Oxford University Press, New York, NY. Hussein, HS & Bollinger, LM 2005, ‘Prevalence of Shiga toxin-producing Escherichia coli in beef cattle’, J. Food Prot. Vol. 68, no. 10, pp. 2224-2241.

Kaper, JB, Nataro, JP & Mobley, HL 2004, ‘Pathogenic E. coli’, Nature Review of Microbiology, vol. 2, no. 2, pp. 123-40.

Karmali, MA 2003, ‘The medical significance of Shiga toxin-producing Escherichia coli infections: An overview’, Methods Mol. Med, vol., 73, pp. 1-7.

13 Latchman, DS 1995, PCR applications in pathology: principles and practice, Oxford University Press, New York, NY. Lazcka, O, Campo, FJ & Munoz, FX 2007, ‘Pathogen detection: a perspective of traditional methods and biosensors’, Biosensors and Bioelectronics, vol. 22, pp. 1205-1217.

Nataro, JP & Kaper, JB 1998, ‘Diarrheagenic Escherichai coli’, Clinical Microbiology Reviews, vol. 11, no. 1, 142-201.

Pelt-Verkuil, E, Belkum, A & Hays, JP 2008, Principles and technical aspects of polymerase chain reaction amplification, Springer, New York, NY. Perelle, S, Dilasser, F, Grout, J & Fach P 2006, ‘Screening food raw materials for the presence of the world’s most frequent clinical cases of Shiga toxin-encoding Escherichia coli O26, O103, O111, O145 and O157’, Int J food Microbiol. vol. 113, 3, pp. 284-288.

Persing, DH 1993, Diagnostic Molecular Microbiology: principles and applications, ASM Press, Rochester.

Sadasivam, S & Manickam, A 1996, Biochemical methods, New Age International, New Delhi.

Smith, JL, Fratamico, PM & Gunther, NW 2007, ‘Review: Extraintestinal pathogenic Escherichia coli’, Foodborne Pathogens and Disease, vol. 4, pp. 134-163.

Wilson, KM, & Walker, JM 2010, Principles and techniques of biochemistry and molecular biology, Cambridge University Press, New York, NY