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All the glassware including test tubes, flasks, beakers, etc, were sterilized in autoclave at 121oC at 15 PSI pressure for 15 minutes. Where needed pre-sterilized disposable forceps, scalpels, etc and Petri-plates and pipettes were used.
3.2 Preparation of Media and Reagents
Various types of media and reagents used for isolation, purification, observation of growth, cultural characteristic, antimicrobial susceptibility testing, PCR and PFGE of E. coli, were prepared according to instructions of the manufacturers. The media were examined for sterility by aerobic incubation at 37oC for 24 hours. The contaminated preparations were discarded by autoclaving at 121oC at 15 PSI pressure. Details of their composition and preparation is mentioned in appendix-4
3.3 Isolation and Identification of Escherichia coli
Sampling plan established by Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) was used (CIPARS, 2006). The plan involved weekly sampling over a period of one year from 19 census divisions in Alberta. The map of the divisions is shown in Fig. 1. During each visit four grocery stores were randomly selected from each of the divisions. The four stores included three chain stores and one independent / butcher store. From each store one sample of each of skin-on chicken leg/wing/breast/thigh, fresh retail ground beef (regular or lean) and turkey drumstick / wing / ground turkey were purchased. A total of three to four samples were collected per store. In this way, total 9 or 12 samples were collected per visit. Each sample was given a unique sample ID, submission ID and project ID number. The information regarding number, name/type, census division and location of the store, number of samples and sampling date were recorded on CIPARS Store Log Worksheet (Annex-1). A total of 433 samples comprising chicken (206), beef (135) and turkey (92) were purchased (Appendix-1). Samples were transported on ice in a cooler to laboratory for isolation of E. coli and their identification (CIPARS, 2006).
3.3.2 Isolation of Escherichia coli
Initial isolation and biochemical characterization of Escherichia coli was performed in collaboration with Agri-Food Research laboratories at Alberta Agriculture, Food and Rural Development. Following procedure was used for isolation of E. coli.
Each of the meat samples was placed on a clean and disinfected surface. Outside of the package was wiped with 70% ethanol in order to its decontamination. Using sterile scissors, the package was opened aseptically. Each of the samples of chicken (one leg, thigh, wing or breast), 25 grams of ground beef and turkey (one wing, breast, drumstick or 25 grams ground turkey) was transferred to a sterilized stomacher bag containing 225 ml of sterilized buffered peptone water (BPW) using sterilized foreceps. Care was taken to avoid contact of external wrapping with the sample. The stomacher bag containing bony meat of either chicken or turkey was shaked vigorously for 15 seconds, allowing to stand for 15 minutes and shaked again for 15 seconds. The stomacher bag containing ground beef or turkey was stomached for 30 seconds at 230 rpm (Stomacher 400 circulator, Seward, UK). Fifty ml of the BPW rinse/dilution of each of the samples was aseptically transferred to a container (whirl pack bag) containing 50 ml double strength E. coli (EC) broth (Fig 2-9).
The BPW-EC broth was incubated at 45oC for 22 hours. With help of 10 ul loop, the BPW-EC broth culture was streaked onto Eosine Methylene blue (EMB) agar and incubated at 35oC for 21 hours. After incubation, the EMB plates were read for any growth without refrigerating the plates. Presence of green metallic sheen with dark center of colony was considered as positive for E. coli.
3.3.3 Biochemical Identification of Escherichia coli
22.214.171.124 Production of gas and presence of florescence
One typical colony showing metallic sheen on EMB plate was transferred to EC broth with MUG (ECM) in test tubes having Durham tubes. The inoculated broth was incubated at 45oC for 48 hours. After incubation, the tubes were examined under 366 nm UV light for any fluorescence and Durham tubes were observed for any gas production. Presence of bright blue fluorescence along with gas production in Durham tubes was considered positive (Feng and Hartman, 1982).
126.96.36.199 Indole test
Indole test was performed using diagnostic slides (BD Diagnostic System, USA) as per manufacturer's instructions. One typical colony showing metallic sheen on EMB plate was smeared onto the reaction area of the slide containing p-Dimethylaminobenzaldehyde reagent. The slide was examined immediately for any color change. Formation of pink color within 30 seconds was considered positive and no change in color was considered negative. E. coli ATCC 25922 and Klebsiella pneumoniae ATCC 13883 were used as positive and negative control, respectively.
188.8.131.52 Preservation of Samples
Two colonies (two isolates) from positive samples were randomly selected and inoculated in TSB. Tubes were incubated at 35oC for 24 hours. After incubation, bacterial suspension was preserved in cryovial at -80oC, by inoculating 300 ul bacterial suspension in 100 ul of glycerol buffer. Each isolate was given a unique isolate number (Appendix 2).
3.4 Confirmation of E. coli by PCR using uspA Primer
The E. coli was confirmed by amplifying universal stress protein (uspA) gene by PCR (Chen and Griffiths, 1998).
3.4.1 Isolation of Bacterial DNA
Isolation of E. coli DNA was made using boiling method (Chen and Griffiths, 1998). The E. coli samples were inoculated in TSB using sterile tooth pick. The TSB containing tubes were incubated at 35oC for 24 hours. After incubation, a loopful culture from each tube was streaked onto pre- labeled TSA plate and incubated at 35oC overnight.
After incubation, using sterile tooth pick, one colony from each isolate was transferred to a pre-labeled PCR strip tubes containing 200 ul of sterile water. The tubes were placed in boiling water bath for 15 minutes. After boiling, the tubes were centrifuged (Eppendorf 5417 C, Eppendorf AG, Hamburg, Germany) at 4000 rpm for 2 minutes. Supernatant containing DNA was separated in pre-labelled PCR tube. The tube was placed at -20oC for further use. Extracted DNA was amplified using uspA specific primers set (Table 1) for confirmation of E. coli.
3.4.2 PCR of E. coli (uspA gene)
PCR were performed by using Taq PCR Master Mix Kit (Qiagen, Mississauga, Ontario, Canada) with 1X Qiagen PCR master mixture, 5 ul of template and 0.5 uM of each primer as per manufacturer's instructions. PCR protocols and primers are described in Table 1. Thermocycler (Mastercycler ep, Eppendorf, Germany) was programmed with standard conditions as mentioned in Table 1a.
3.4.3 Gel Electrophoresis
After polymerization, agarose gel was placed in electrophoresis tank (Sub-Cell Model 96 Cell, Horizontal, Bio-Rad Laboratories, Inc. Hercules, CA, USA), containing TBE buffer, and loaded with PCR product, positive and negative control and 100 bp ladder. Voltage was adjusted to 110 volts for 90 minutes. The gel was stained with Ethidium bromide solution for 20 minutes and visualized under UV light using Kodak Gel Logic 100 Imaging system (Eastman Kodak Company, New Haven, CT, USA).
3.5 Antimicrobial Susceptibility Testing
Antimicrobial susceptibility of E. coli isolates from various retail meat samples was tested using automated microbroth dilution method. The minimal inhibitory concentrations (MIC) were determined using a sensititre system (Trek Diagnostic Systems, Ohio, USA). The results were interpreted according to Clinical Laboratory Standard Institute (CLSI) guidelines. The isolates having MICs greater than resistance breakpoint were considered as "resistant" (Table -2). Isolates with intermediate MICs were considered susceptible (CLSI, 2010).
E. coli isolates and quality control (QC) organism (E. coli ATCC 25922) were cultured onto MacConkey agar plate while other quality control organisms (Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213) were cultured onto Muller Hinton agar and Enterococcus faecalis ATCC 29212 was cultured onto Blood agar plates (BAP) (Oxoid). Plates were incubated at 35oC for 18 hours. One colony of test stain and QC organisms were selected and sub-cultured onto Muller Hinton agar plates and incubated at 35oC 22 hours. After incubation, a small portion of growth was transferred to 5 ml pre-sterilized demineralized water (Trek Diagnostic Systems, Ohio, USA) using sterile cotton tip applicator (Puritan, USA). Sample was vortexed (Vortex genie, Fisher Scientific) and was placed in already calibrated (using 0.5 McFarland Standard) Nephlometer of auto-inoculator (Trek Diagnostic Systems, Ohio, USA). Nephlometer reading of sample was adjusted in green zone by either adding demineralized water drop-wise or more bacterial cells.
Ten ul of bacterial suspension was transferred to 11 ml of Mueller Hinton broth (Trek Diagnostic Systems, Ohio, USA) using a calibrated loop (Simport, Vietnam). The broth was mixed well. After applying dose head onto the tube, it was placed into autoinoculater and 50 ul of broth was transferred into each well of custom plates (CMV1AGNF) for Gram negative bacteria as used by CIPARS (plate format in the Fig 10). Each plate was covered with sticky plastic cover and labelled on side and then incubated at 35oC for 18 hours while QC plate of Enterococcus faecalis ATCC 29212 was incubated at the same temperature for 24 hours. The plates then read using autoreader and SWIN software (Trek Diagnostic Systems, Ohio, USA) (Fig 11 & 12). The QC organisms were tested once a month and with each batch of samples E. coli ATCC 25922 was also run as a quality control organism.
3.6 Detection of Antimicrobial Resistance Genes
Bacterial DNA of each isolate was extracted by the techniques as described in Section 3.4.1. Antimicrobial resistant genes in E. coli isolated from various retail meat samples were determined using PCR. Genotype of an isolate was considered as "positive" if it carried a gene encoding for resistance to a particular drug. Major antimicrobial resistance genes for tetracycline (tetA, tetB and tetC), sulfonamides (sul1, sul2 and sul3), Î²-lactamase (blaCMY-2, blaSHV and blaTEM), Kanamycin (aphA1, aphA2 and aadB), gentamicin (aadB and aac3 (IV)), streptomycin (strA and strB and aadA) were amplified using a multiplex PCR in collaboration with Department of Pathology, University of Guelph, Ontario, Canada. The procedure used for PCR has been described by Gosia et al. (2009).
3.6.1 PCR of Antimicrobial Resistance Genes
PCR were performed by using Qiagen multiplex PCR kit (Qiagen, Mississauga, Ontario, Canada) with 1X Qiagen multiplex PCR master mixture, 1X Q-solution, and 1X primer mixture according to the manufacturer's instructions. PCR protocols and primers are described in Table 3 & 4. Three pairs of primers were used in each multiplex PCR, as shown in Table 3. Thermocycler (Mastercycler ep, Eppendorf, Germany) was programmed with standard conditions as mentioned in Table 4. The gel electrophoresis, staining and documentation was performed as described in Section 3.4.3
3.7 DNA Fingerprinting of E. coli Isolates
Genomic DNA fingerprinting patterns of E. coli was determined using Pulsed-Field Gel Electrophoresis (PFGE) method as described by CDC (2004).
3.7.1 Preparation of PFGE Plugs
Frozen E. coli isolates and Salmonella braenderup (H9812) as control in glycerol buffer were cultured in pre-labelled and pre-sterilized TSB tubes and incubated at 37oC for 18 hours. The bacterial culture was transferred onto TSA plate using sterile cotton swab, for confluent growth (Fig 13& 14) and incubated at 35oC for 18 hours. Care was taken to avoid prolong incubation (more than 18 hours).
Five ml Cell Suspension Buffer (CSB) was dispensed in pre-sterilized test tubes. Using sterile cotton swab, the bacterial growth was transferred to CSB solution (Fig 15). The tubes were mixed to make uniform suspension. Spectrophotometer (Cell density meter 40, Fisher Scientific) was set to zero with wavelength 600 nm using blank CSB solution and then sample tubes (CSB) were placed in densitometer to determine the optical density (OD). The OD was adjusted in between 1.3 to 1.4 (average 1.35) (Fig 16) either by adding more CSB or bacterial cell. The bacterial cell suspension (500 ul) was transferred to a pre-labeled 1.5 ml microfuge tube. 25 ul proteinase K (PK) was added and mixed gently by inverting 6 times. Molten low melting point (LMP) agarose (Ultrapure LMP agarose, Invitrogen, Mississauga Ontario, Canada) (500 ul) was added to cell suspension-PK mixture and mixed gently by inverting 6 times. Using micropipette 100 ul of cell suspension-PK-agarose mixture was dispensed immediately into pre-labeled plug molds (Fig 17) avoiding creation of air bubbles. The mold was placed at room temperature for about 10 minutes for solidifying the agarose plugs.
3.7.2 Cell Lysis
Two ml sterilized microfuge tubes were labeled and 1.5 ml cell lysis-PK solution was dispensed into each of the tube. After solidification the plugs were inserted in the microfuge tube (Fig 18). The tubes were placed in floatie rack and incubated in shaking water bath (Julabo SW 23, Allentown, PA, USA) at 54oC for 2 hours with speed setting ~10%.
3.7.3 Slice washing
The tubes containing the plugs were removed from water bath. Individual plug was transferred on clean parafilm (Fig 19). Using clean single edged razor, 2 mm slice from plug was cut (Fig 20) and dispensed into pre- labeled microfuge tubes. Sterile type 1 water (750 ul) was added to wash slices in the tube and allowed to stand for 15 minutes. After 15 minutes, water from microfuge tube was removed using vacuum pump and slice was washed again with sterile type 1 water. Washing was repeated three more times with 750 ul TE solution. Finally TE was removed and 750 ul REact 2 (Invitrogen, Mississauga Ontario, Canada) was added in each slice and allowed to stand for 15 minutes.
3.7.4 DNA Restriction Digestion
The REact 2 was removed and 200 ul XbaI master-mix was added in each slice. It was ensured that slices were submerged in the solution. Slices were incubated at 37oC for 2 hours.
3.7.5 Gel casting and CHEF preparation
One gram ultrapure agarose (Ultrapure agarose, Invitrogen, Mississauga Ontario, Canada) was weighed and dissolved in 100 ml of 0.5 X TBE. Agarose was melted using microwave oven and allowed to stand at 55oC. After incubation, slices were removed from incubator. The XbaI master mix was removed and 500 ul 0.5 X TBE was added in each microfuge tube and incubated for 5 minutes at room temperature. After incubation, slices were removed from 0.5 X TBE and were aligned on comb (Fig 21) along with Salmonella braenderup (H9812) as standard. The sequence of samples on comb was recorded on PFGE worksheet (Annex 2). Slices were allowed to dry for 5 minutes. Casting stand was assembled and 20 well comb was inserted into casting stand. Molten agarose (1 %) was gently poured into the casting stand and allowed to solidify for 30 minutes (Fig 22). Electrophoresis chamber was filled with freshly prepared 0.5 X TBE solution (2000 ml) and CHEF-DR III (Bio-Rad Laboratories, Inc. Hercules, CA, USA) was adjusted to following setting and was turned on (Fig 23 & 24)
Water pump of CHEF-DR III was turned on. Once buffer started flowing through rubber tubing, attached chiller was turned on and temperature adjusted to 14oC. When temperature of buffer in electrophoresis chamber was reached to 14oC, comb from the gel was removed gently. The gel with its platform was removed from casting stand and inserted into the casting platform of electrophoresis chamber. Chamber lid was closed and power supply was turned on.
3.7.6. Staining and documentation of gel
After 18 hours electrophoresis, gel was removed from chamber and stained with ethidium bromide solution for 20 minutes and visualized under UV light using Kodak Gel Logic 100 system. The image was saved as Tagged Image Format File (TIFF) for further analysis.
3.7.7 Analysis of PFGE patterns
DNA patterns obtained with the PFGE techniques were analyzed with Bionumeric 6.0 software (Applied-Maths, Ghent, Belgium) after conversion and normalisation. Similarities between the DNA patterns based on band positions were determined from the Dice similarity coefficients calculated by software. The PFGE patterns with Dice similarity coefficients greater than 90 percent were considered genetically related and were assigned as PFGE type. A dendogram was constructed by using the unweighted pair group method with arithmetic averages to reflect similarities in the matrix. The interpretation was made as described by Tenover et al. (1995).
3.8 Statistical Analysis
The MIC values and antimicrobial resistance gene data was maintained using Microsoft Excel (MS Excel 2003, Microsoft Corporation, Redmond, WA). Descriptive Statistical analysis including percentages, confidence interval and graphs were produced using commercially available statistical package SPSS (version 18.0, for Windows; SPSS, Chicago, IL). Statistical Association of antimicrobial resistant E. coli with retail meat types, store type, season of the year, antimicrobial resistant genes and antimicrobial resistance was estimated by Fisher Exact Test using SAS (version 9.2, for windows, SAS Institute, Cary, NC) as described by Rosengren et al. (2009).