Antibiotic Sensitivity and Evaluation of Plasmid Profile of Major Foodborne Pathogens

Antibiotics Susceptibility Test

Susceptibility of isolates to different antibiotics were tested following Kirby Bauer disc diffusion method using Muller Hinton Agar against selected antibiotics, namely Ampicillin (25μg), Chloramphenicol (50μg), Kanamycin (30μg), Streptomycin (30μg) and Tetracycline (100μg) (Hi-Media, Mumbai). Amoxicillin(30μg), Amoxicillin-Clavulanic acid (30 μg), Cefoxitin (30 μg), Cefotaxime (30 μg), Ceftriaxone (30 μg), Cefuroxime (30 μg), Ceftazidime (30 μg), Gentamicin (10 μg),), Ofloxacin (30 μg), Cloxacillin (5μg), Nitrofurantoin (30 μg) Levofloxacin (30 μg), Ciprofloxacin (30 μg) (Oxoid, UK). Inhibition zone size was interpreted using standard recommendation of National Committee for Clinical Laboratory Standards [N.C.C.L., 2000] now known as Clinical Laboratory Standard Institute (CLSI) [C.L.S.I., 2005]

Procedure For Performing The Disc Diffusion Test

The growth method was performed as follow;

(1) At least three to five well isolated colonies of the same morphological type was selected from an agar plate culture. The top of each was touched with a loop, and the growth was transferred into a tube containing 4 to 5ml of a suitable broth medium.(2) The broth culture was incubated until it achieved or exceeded the turbidity of the 0.5McFarland standards which took up to six hours.

Inoculation of Test Plates.

Optimally, within 15 minutes after adjusting the turbidity of the inoculum's suspension, a sterile cotton swab was dipped into the suspension. The swab was then rotated several times and pressed firmly on the inside wall of the tube above fluid level. This was done to remove excess inoculums from the swab.(2) The dried surface of a Mueller-Hinton agar was inoculated by streaking the swab over the entire sterile agar surface. This procedure was repeated by streaking two or more times, by rotating the plate approximately 60^?C each time to ensure an even distribution of inoculums. As a final step. the rim of the agar was swabbed.(3) The lid was left ajar for some minutes to allow for any excess surface moisture to be absorbed before applying the drug-impregnated discs.

Placement of Discs

The predetermined battery of antimicrobial discs was dispensed onto the surface of the inoculated agar plate. Each disc was pressed down to ensure complete contact with the agar surface. Whether individually or with a dispensing apparatus, they must be distributed evenly so that they are no close than 24mm from center to center. Ordinarily, no more than 12 discs should be placed on 150mm plate, or more than five discs on a 100m plate. Because some of the drug diffuses almost instantaneously, a disc should not be relocated once it has come into contact with the agar surface. Instead, place a new disc in another location on the agar.

The plates were then inverted and placed in an incubator set to 350c within 15 minutes after the discs are applied.

Reading Plates and interpreting results

After 16 to 18 hours of incubation, each plate was examined, the diameters of the zones of inhibition (as judged by the unaided eye) are measured, including the diameter of the disc.

Zones are measured to the nearest whole millimeter, using sliding calipers or a ruler, which is held on the back of the inverted Petri plate. The sizes of the zone of inhibition are interpreted by referring to the NCCL's table on Zone Diameter Interpretive Standards and Equivalent Minimal Inhibitory Concentration (MIC) Break points for Enterobacteriaceae.

Plasmid Profile Analysis

Plasmids are circular deoxyribonucleic acid molecules that exist in bacteria, usually independent of the chromosome. The study of plasmids is important to medical microbiology because plasmids can encode genes for antibiotic resistance or virulence factors. Plasmids can also serve as markers of various bacterial strains when a typing system referred to as plasmid profiling, or plasmid fingerprinting is used. In these methods, partially purified deoxyribonucleic acid species are separated according to molecular size by agarose gel electrophoresis. In a second procedure, plasmid deoxyribonucleic acid which has been cleared by restriction endonucleases can be separated by agarose gel electrophoresis and the resulting pattern of fragments was used to verify the identity of bacterial isolates. Because of the many species of bacteria, plasmid profile analysis has been used to investigate outbreaks of many bacterial diseases and to trace inter-and intra species spread of antibiotic resistance.

Mechanism of plasmid profile analysis

Plasmids were detected by a modified rapid method. Cells were grown in 3ml of L-broth overnight at 30°C to an optical density at 600mm of 0.8 and pelleted by centrifugation (5,700 rpm, 4°C, for 7 min in a Sorvall SS34 rotor). The cell pellets were thoroughly suspended in 1ml of E buffer. The cells were then lysed by adding 2ml of lysing solution, which was mixed by brief agitation.

The solution was heated at 50°C to 65°C for 20min in a water bath, and 2ml volumes of phenolcholoform solution (1:1, vol/vol) were added. The solution was emulsified by shaking, and the emulsion was broken by centrifugation (6,000 rpm, 15min, 4°c IN A Sorvall SS34 rotor). Avoiding the precipitate at the interface, the upper aqueous phase was transferred to a screw cap tube by using a polyethylene Pasteur pipette (Ulster Scientific, Inc, Highland, N.Y). Sample was withdrawn directly for electrophoresis immediately. 35pl of sample was mixed on a square of parafilm “M” (American Can Company, Greenwich, Conn.) with 10 pl of 0.25% bromocresol purple in 50% glycerol-0.05 M Tris- acetate (pH7.9). To minimize sharing of large plasmids it is important that samples be withdrawn and transferred with Drummond 50-ud micropipettes and an Adams no 4555 suction apparatus (Becton, Dickinson & Co., Rutherford, N.J. ) After centrifugation, the supernatant was subjected to agarose gel electrophoresis for the detection and sizing of plasmid DNA.

The organism were separated into two groups (i.e gram positive and gram negative) and the extraction mechanism for each was applied. Details are given below;

Extraction of plasmids from Gram positive bacteria

The plasmids were extracted by growing the bacterial cellsovernight on Luria -Bertani medium supplemented with 50μg/mL ampicillin. It was then centrifuged at 13,000 rpm for 2 minutes to pellet cells. 500μL of normal saline was added, vortex and pulse at 13,000 rpm for 10 seconds to wash cells. The supernatantwas removed and cells resuspended in 500μL of Tris (10mM)-EDTA (1mM)-Sucrose (50mM) buffer (pH 8.0). Later 500μL Lysis buffer solution containing SDS (1%) and Lysozyme (2mg /mL) in Tris (10mM)- EDTA (1mM) buffer (pH 8.0) added. Then incubated at 37°C for 2 hours. The preparation was boiled at 95°C for 3 minutes and the suspension was neutralized by adding 3M KOAC (pH 5.2). Then the suspension wascentrifuged at 13,000 rpm for 5 minutes. The upper layer was transfered into a new sterile Eppendorf tube. Twice the volume of absolute ethanol (95.5%) was added and centrifuged at 10,000 rpm for 5 minutes. The supernatant was decanted and pellet washed with 70% ethanol. Twice the volume of absolute ethanal was added and centrifuged and the pellet was dried in the air. The DNA pellet was re-dissolved in 40μL of Tris-EDTA buffer (pH 8.0). Then the purity of the DNA solution was assessed using nanodrop Spectrophotometer and the plasmid DNA samples were electrophoresed on 0.9% agarose at 70V for 40 min.

Plasmid DNA Isolation from Gram Negative Bacteria (Alkaline Lysis method of Bimboimand Doly, 1979)

200μl of buffer 1A was added to cell pellet and vortex, and 400μl lysing solution. The test tubes were inverted 20 times at room temperature. 300μl of ice cold buffer 2B added, vortex and keep on ice for 30 minutes. Suspension centrifuged at 3,000xg for 15 minutes. To the supernatant, 700μl of chloroform was added and Vortex then centrifuged at 3,000xg for 10 minutes. To 500μl aqueous layer, 1ml of absolute ethanol was added and kept in ice for 1 hour, and again centrifuged at 3000xg for 30 minutes. Pellet was then washed with 70% ethanol, decanted and dried. Then 50μl of buffer 3C was added to the pellet.

The composition of each buffer solution and lysing solution are given below;

Plasmid DNA was extracted by alkaline lysis method with (Gram +ve) and without (Gram −ve) Lysozyme. Electrophoresis was done horizontally on 1% agarose gel at 70V for 40 minutes, stained with ethidium bromide, visualized under UV light and photographed thereafter.

Sizes of DNAs were extrapolated based on the relative mobilities of 10kb ladder DNA markers that were co-electrophoresed with the DNA samples.