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A total number of 20 consecutive non-repetitive clinical isolates of Escherichia coli and 20 non-repetitive clinical isolates of Klebsiella pneumoniae were obtained from UTI patients from a tertiary care hospital in Coimbatore. Urine cultures were collected over a period, from June-September 2010, and were subjected to identification and characterization by performing Gram staining, motility and biochemical tests as per standard protocol.
Antimicrobial susceptibility testing were done with various antibiotic standard discs including Pencillin and related drugs, Cephalosporin drugs, Cephamycins (cefoxitin) and Carbapenems (meropenem) using standard Kirby-Bauer disc diffusion method.
Screening for AmpC producing strain by modified-disk based method using boronic acid as an inhibitor for AmpC positive isolates on Mueller-Hinton agar plates, was performed by Kirby-Bauer disc diffusion method of the resistance antibiotic strains.
The isolates found as plasmid-mediated AmpC positive strains, based on modified-disk based phenotypic results, were evaluated for plasmid DNA isolation and AmpC determination by PCR amplification techniques followed by DNA fingerprinting analysis (RFLP) of urine cultures on agarose gel electrophoresis. The DNA bands on the gel were then visualized under UV transilluminator and recorded.
Isolation and Identification of E.coli and K. pneumoniae isolates from urine cultures
A total number of 20 non-repetitive clinical isolates of Escherichia coli and 20 non-repetitive clinical isolates of Klebsiella pneumoniae were obtained from UTI patients collected in Mueller-Hinton agar slants were stored in refrigerator at 4Â°C.
Lactose fermenting colonies on EMB Agar and Mac - Conkey Agar with significant bacteriuria were processed and identified as E.coli and Klebsiella pneumoniae by performing Gram-staining, motility test, colony morphology and also by performing standard biochemical tests - IMViC, Test for H2S production and carbohydate fermentation (glucose, lactose, mannitol and sucrose) as per standard protocol.
1. Morphological and biochemical characterization of clinical isolates for the following tests,
a. Gram staining
Using aseptic techniques, a smear was prepared using a clean, dry, grease-free glass slide by an inoculating loop. The smear was allowed to air-dry and then heat fixed. The smear was flooded with crystal violet and allowed to stand for
1 minute and then washed with tap water. The slide was then flooded with Gram's iodine as mordant and allowed to stand for a minute. The smear was then washed with tap water. Decolorisation was done using 95% alcohol and the slide was washed again with tap water. It was then counterstained with safranin for 45 seconds and washed again with tap water. Finally, the slide was blotted dry with bibulous paper and examined under oil immersion.
b. Motility by using Hanging-Drop method
Using aseptic techniques, a thin layer of petroleum jelly was applied along the edges of a cover slip with a cotton swab. A loopful of culture was then placed in the center of a cover slip. The clean glass depression slide was placed over the cover slip with the concave surface facing down, to avoid the drop of culture touching over the depression slide. The slide was gently pressed to fix firmly with the cover slip and inverted so that the drop continued to adhered to the inner surface of the cover slip. Then the slide was examined under the microscope by focusing the edge of the drop culture under the low power objective and finally reducing the light source using condenser and observing under high power objective.
c. Biochemical tests
i. Indole test:
Using aseptic techniques, each organism was inoculated into appropriately labeled SIM agar deep tubes by stab inoculation. The inoculated tubes were incubated for 24 to 48 hrs at 37Â°C. The incubated test tubes were gently removed and 10 drops of Kovac's reagent was added. Colour obtained in the test tube was examined and recorded. One tube served as a control.
ii. Methyl Red test:
Using aseptic techniques, each organism was inoculated into appropriately labeled MRVP broth tubes by a loop inoculation. The inoculated tubes were incubated for 24 to 48 hrs at 37Â°C. The incubated test tubes were gently removed and 5 drops of methyl red indicator was added. Colour obtained in the test tube was examined and recorded. One tube served as a control.
iii. Voges-Proskauer test:
Using aseptic techniques, each organism was inoculated into appropriately labeled MRVP broth tubes by a loop inoculation. The inoculated tubes were incubated for 24 to 48 hrs at 37Â°C. The incubated test tubes were gently removed and 10 drops of Barritt's reagent (A) was added and the tubes were shaken well. Immediately, 10 drops of Barritt's reagent (B) was added and shaken well. The culture tubes were slightly shaken every 2-3 minute, and the colour obtained in the test tube was examined and recorded. One tube served as a control.
iv. Citrate utilization test:
Using aseptic techniques, each organism was inoculated into appropriately labeled Simmon's citrate agar slants tube by streak inoculation. The inoculated tubes were incubated for 24 to 48 hrs at 37Â°C. The agar slant was examined for change in the colour of the medium from green to prussian blue. One tube served as a control.
v. Hydrogen sulphide test:
Using aseptic techniques, each organism was inoculated into appropriately labeled SIM agar deep tubes by stab inoculation. The inoculated tubes were incubated for 24 to 48 hrs at 37Â°C. The tubes were examined for the presence or absence of black colouration along the line of stab inoculation and the result was recorded. One tube served as a control
vi. Carbohydrate fermentation:
Using aseptic technique, each organism was inoculated into appropriately labeled fermentation broth tubes containing phenol red lactose, sucrose, glucose and mannitol broths along with Durham tubes. The inoculated tubes were incubated for 24 to 48 hrs at 37Â°C. All carbohydrate broth cultures were examined for change in colour and presence or absence of a gas bubble was observed and recorded. One tube served as a control (Cappuccino and Sherman., 2009)
Preservation of standardized Cultures
The identified and characterized urinary isolate were sub-cultured and preserved at 4Â°C.
2. Preparation of Mc. Farlands Standard.
Preparation of 0.5 Mc. Farlands standard by mixing 0.5 ml of 0.048 M Bacl2 (1.175% w/v Barium chloride dehydrate ) with 99.5 ml of 0.35 N H2SO4 (1% v/v). This produced an inoculating suspension of approximately 108 CFU per ml, which was then diluted in fresh broth to final inocula of approximately 105 CFU/ml. The standardized bacterial cultures were used for the other tests, which were done in triplicates (Victor lorain).
The clinical isolates were pre-cultured in Mueller Hinton broth and incubated at 37Â°C for 24 hrs. The inoculum was standardized by matching the turbidity of the clinical cultures visually to 0.5 Mc. Farlands standard.
3. Antibiotic susceptibility testing were performed by Kirby-Bauer disc diffusion method
Antibiotic susceptibility testing was done to select the resistant strains of the 20 consecutive non-repetitive clinical isolates of Escherichia coli & 20 isolates of Klebsiella pneumoniae were carried out on Mueller-Hinton agar plates by Kirby-Bauer disc diffusion technique.
Susceptibility of clinical isolates was tested with various antibiotics standard disc for the following drugs, Ampicillin (10Âµg/disc), Amoxycillin (10Âµg/disc), Penicillin G (10Âµg/disc), Ofloxacin (5Âµg/disc), Ciprofloxacin (5Âµg/disc), Cefixime (5Âµg/disc), Cefpodoxime (10Âµg/disc), Cefazolin (30Âµg/disc), Cefpirome (30Âµg/disc), Cephalothin (30Âµg/disc), Cefepime (30Âµg/disc), Cephalexin (30Âµg/disc), Cefotaxim (30Âµg/disc), Cefotetan (30Âµg/disc), Cephadroxil (30Âµg/disc), Ceftazidime (30Âµg/disc), Ceftriaxone (30Âµg/disc), Cefuroxime (30Âµg/disc), Ceftrizoxime (30Âµg/disc), Cefoxitin (30Âµg/disc), Meropenem (10Âµg/disc) Piperacillin (100Âµg/disc).
The characterized UTI clinical isolates were inoculated on the previously prepared Mueller-Hinton agar plates by using a sterile cotton swab. The standard antibiotic discs were then placed on the inoculated media and kept for 15 minutes for diffusion. The plates were finally incubated for 24 hours at 37Â°C and zone of inhibition was observed and recorded by antibiotic zone scale. .
4. Screening test for AmpC Î²- lactamase using modified-disc method by Cefoxitin-phenyl boronic acid
Preparation of Cefoxitin-phenyl boronic acid disc:
The stock solution of 120 mg phenylboronic acid was dissolved in 3ml of dimethyl sulphoxide and 3ml of sterile distilled water was added to this solution. Pipette out 20Î¼l of phenyl boronic acid (400Î¼g) from the prepared stock solution and dispensed onto cefoxitin 30Î¼g disc. Disc was allowed to dry for 20-30 minutes. (Wookeun Lee et al., 2009).
Method for AmpC detection:
Over night culture of clinical isolates was swabbed with sterile cotton swab on Mueller-Hinton agar plates. Disc susceptibility testing was performed by antibiotic disc containing cefoxitin 30Î¼g and a similar disc containing cefoxitin 30Î¼g supplemented with 400Î¼g of phenyl boronic acid were placed on the swabbed agar plates at a distance of 30mm and kept aside for 10-15mts for diffusion. Incubate the plate at 37Â°C for 24 hrs, zone of inhibition was observed and measured with antibiotic zone scale.
5. Genetic evaluation of selected clinical isolates by
a. Isolation of plasmid DNA from bacterial cells,
Plasmid DNA was isolation from the bacterial cells (clinical strains) was done by alkaline lysis method. The selected resistance strains were transferred in aseptic hood from Mueller-Hinton agar slants to Luria Bertani agar slants and stored at 4Â°C in refrigerator to preserve the master culture. Simultaneously, they were inoculated in 10ml LB broth with antibiotic and grown over night at 37Â°C.
After 24 hrs of incubation, 2 ml of saturated bacterial culture was pipetted in 4ml centrifuge tubes, spun at 10,000 rpm for 1 minute and the supernatant solution was discarded. Again the process was repeated once to recover more number of cell pellets. To the cell pellets add 0.2ml of ice cold solution I (Glucose, Tris Hcl (pH 8.0) & EDTA (pH 8.0) and re-suspend the cells thoroughly. Again add 0.4ml solution II (1% SDS & 0.2 N NaOH) to this and invert gently 5 times. Keep the centrifuge tubes aside at room temperature for 5 minutes. Then add 0.3 ml of ice-cold solution III (Potassium acetate & Glacial acetic acid) and again inverting the tubes gently for 5 times, cool the tubes for 10 minutes on the ice-cold gel pack.
Centrifuge the tubes for 5 minutes. Transfer the supernatant to fresh 4 ml centrifuge tubes using clean micropipette tips. Leave of the white precipitate while transferring the supernatant solution. Fill the remaining volume of the centrifuge tubes with isoproponal and keep it at room temperature for 2 minutes. Again centrifuge the tube once for 5 minutes and decant the supernatant solution from the centrifuge tube without dumping the milky white pellet. Finally, add 1 ml of ice-cold 70% ethanol, invert the centrifuge tube several times and spin the tubes for 1 minute. Taking care, decant the supernatant without dumping out the pellets. Air-dry the pellets in the centrifuge tubes till the ethanol evaporates. Add 50Î¼l of TE buffer (tris-EDTA) to all isolated tubes. Centrifuge the tube to re-suspend the white pellets completely in the buffer solution. The isolated plasmid DNA obtained from the bacterial cells was kept at room temperature for 5 minutes before storing at -20Â°C till it requires.
Weigh 0.45 grams 1.8% agarose in a clean 50ml beaker. To this add 0.5 ml 50X TAE buffer and 24.5 ml distilled water and heat it till the agar melts to form a clear solution. Then add a pinch of ethidium bromide to the clear solution and solidify it in the gel tray with required combs (comb type-8, 5 and 4).
From the 50Î¼l of isolated plasmid DNA, 10Î¼l was loaded to the agarose well with 5Î¼l of 6X gel loading buffer and run by electrophoresis unit at 50-100 volts for 2-3 hours till the tracking dye reached 3/4th length of the gel. The DNA fragment was then visualized under UV transilluminator (Alpha digidoc) and photo images are recorded.
b. AmpC determination by PCR amplification techniques.
AmpC Primer Sequences.
PCR reaction was performed in PCR tubes, 50Î¼l as final volume.
Thaw the primers, nuclease free water and isolated plasmid DNA are kept aside on ice-cold gel pack for the PCR reactions.
In a fresh PCR tubes add 25Î¼l of mater mix and then add 2Î¼l of forward primer and 2Î¼l of reverse primer. To this mix add 2Î¼l of plasmid DNA and 19Î¼l of nuclease free water as final volume to 50Î¼l. Prepared 50Î¼l PCR sample was then centrifuged with mini-spin for proper mixing and reaction were carried out by PCR amplification technique for the following thermo cycle processes,
EVENT 1: (Cycle 2 steps-1 repeats) -Initial denaturation step,
Time- 1minute 20 sec
Time-1 minute 20 sec
EVENT 2: (Cycle 3 steps-25 repeats)-Amplification step,
EVENT 3: (Cycle 4 steps-1 repeats)-Final extension,
Time- 1minute 20 sec
Time-1minute 20 sec
Time-1minute 20 sec
EVENT 4: (Hold)
Hold in time for 60 sec.
After PCR amplification, 15Î¼l of PCR amplified product with 5Î¼l of 6Xgel loading buffer was loaded to the 2% agarose well stained with ethidium bromide. The amplified product was performed using electrophoresis unit at 50-100 volts for 2-3 hrs until the tracking dye reaches 3/4th length of gel. Interpreted of amplified product was compared with DNA ladder on the gel visualized under UV transilluminator (Alpha digidoc) and photo images are recorded. The remaining PCR amplified product was stored at -20Â°C until it requires for sequencing.
6. Identification of DNA fingerprinting by RFLP analysis.
Thaw the vial containing the restriction enzymes, plasmid DNA, nuclease free water and 10X restriction buffer and keep ready aside over the ice cold gel pack for the restriction digestion reaction.
The centrifuge tubes were labeled with the respective names of different samples and 33Î¼l of nuclease free water was added to each tube, followed by 5Î¼l of 10 x restriction buffer was added to all the tubes. Then 10Î¼l of isolated plasmid DNA sample was added to the mixture, followed by 2Î¼l of restriction enzymes. Spin briefly so the content in the centrifuge tubes mixes well by gently tapping and the tubes were incubated at 37Â°C for 2 hrs.
After 2 hrs of restriction digestion, 1.8% gel was prepared using agarose and 1X TAE buffer with a pinch of ethidium bromide. Loaded 5Î¼l of gel loading buffer with 10Î¼l of digested samples run using electrophoresis unit at 50-100 volts for 2-4 hrs till the tracking dye reached 3/4th length of the gel. The restriction fragments on the gel were then visualized under UV transilluminator (Alpha digidoc) and photo images are recorded.