sensitivity pattern of Escherichia

Aim: The study was undertaken to isolate pathogenic E. coli from milk and various milk products, detection of virulence gene using Polymerase chain reaction (PCR) and investigate their antibiotic sensitivity pattern. 
 
Materials and Methods: Altogether 250 milk and various milk products samples consisting of raw milk (50), cheese (50), ice-cream (50), mawa (50) and dahi (50) were collected from milk vendors, retail shops located in Anand city, under aseptic precautions. For the enrichment of the organism from the collected samples, MacConkey broth was used and inoculation was carried out on MacConkey agar and EMB agar. Later on, to confirm the isolates, various biochemical tests such as IMViC test, Urease test were performed. Evaluation of antibiotic sensitivity pattern of E. coli was assessed by disk diffusion method. Finally the E. coli isolates were screened for the presence of virulence associated genes by PCR . 
 
Results: The prevalence of E. coli was observed 32 % in the samples comprising of milk (52.00%), cheese (28.00%), icecream (20.00%), mawa (44.00%), and dahi (16.00%). Antibiotic sensitivity was recorded high for Co-trimoxazole (100%) followed by Gentamicin (96.73%), Trimithoprime (93.47%) and Doxycycline hydochloride (92.39%). Least sensitivity was recorded for Ampicillin (8.69%). In this study, out of 80 E. coli isolates, 25 isolates (31.25%) were positive for stx genes, of which 7 (8.75%) isolates were positive for stx1 gene only, while 12 (15.00%) isolates were positive for stx2 gene only and 5 (6.25%) isolates were positive for both stx1 and stx2, 7 isolates (8.75%) were positive for eaeA gene and all the isolate were negetive for rfb O157 gene. 
 
Conclusions: Current study supports the finding that raw milk and various milk products can be regarded as critical source of pathogenic E. coli This explains the need of strict monitoring and surveillance for effective measures of hygiene and sanitary practice during production of milk and various milk products.


Introduction
The most important causes of food borne diseases are Shiga toxin-producing E. coli (STEC) among the Raw milk consumers have existed in various parts other seropathotypes of E. coli [5].STEC produce of the world.World milk production reached 724 various complications including diarrhea, hemolytic million tons in 2010 as per Food and Agriculture uremic syndrome (HUS) and hemorrhagic colitis (HC) Organization, resulting in trade and massive [6].Report indicate that consumption of raw milk and consumption of various milk products [1].Raw milk is various milk products related with occurrence of 1 to5 consumed directly by a large population in rural areas.
per cent of food infections and among that 53 per cent Milk is an excellent medium for the growth of of cases produced by enteropathogenic E. coli (EPEC) [7].numerous microbes which produce consequential In view of the these particulars, the current study spoilage of the milk and various milk products or was undertaken to detect and characterize the E. coli infections in consumers [2].Because of the specific from milk and various milk products, collected from production, it is impossible to avoid contamination of the milk vendors, retail shops in and around Anand city, milk with microorganisms therefore the microbial Gujarat.content of milk is a major feature in determining its quality [3].The existence of food borne pathogens in Health department in an ice box for further processing removal of cell debris and 3 ìl of the supernatant was and microbiological analysis.
used as a DNA template in PCR reaction mixture.
Isolation and identification: Samples were processed Polymerase chain reaction (PCR): All the E. coli to isolate the E. coli as per the standard Bacteriological isolates were first screened for the presence of virulence associated genes by using the PCR technique Analytical Manual (BAM), U.S. Food and Drug for the detection of different genes.The PCR was Administration (USFDA) method [8].The samples standardized for the detection stx1, stx2, eaeA and rfb were enrichmed in MacConkey broth, the loopful of O157 following the methodology as described by culture inoculates MacConkey agar.Pink colour Paneto et al. [11] for detection of stx1 & stx2 genes, Elcolonies obtain from MacConkey agar were taken and Jakee et al. [12] for detection of eaeA gene and inoculate on Eosin methelene blue agar.Greenish Dhanashee and Mallaya [13] for detection of rfbO157 metallic sheen colonies obtain on EMB agar were with suitable modifications ( The antibiotic susceptibility gel loading buffer along with 5 µl of the PCR product, tests were performed as per method described by Bauer then electrophoresis was performed with use of DNA et al. [10] to find out the antibiotic sensitivity of E. coli.molecular weight marker (Gene Ruler, MBI Fermentas).In vitro antibiotic sensitivity test of the E. coli isolates Agarose gel (2%) along with ethidium bromide (at the was conducted by paper disc diffusion method using rate of 0.5 µg/ml) was used.Electrophoresis was the discs supplied by HiMedia Laboratories Pvt. Ltd., performed in 0.5X TBE buffer at 5V/cm for 60 min.Mumbai (India).Antibiotics used in this test viz.
Prevalence of E. coli: The prevalence of E. coli was observed 32% cent in the samples comprising of milk DNA isolation: The DNA of E. coli isolates was (52.00%), cheese (28.00%), ice-cream (20.00%), mawa prepared by using boiling method.First 100 ìl of (44.00%), and dahi (16.00%).sterilized DNAse and RNAse free water was taken in micro centrifuge tube and approximately loopful of Prevalence of antibiotic sensitivity of E. coli: In present culture was added.Then denaturation was carried out investigation among 80 E. coli isolates from milk and at 95ºC for 10 min, centrifugation was done for the various milk products the highest sensitivity was ice-cream was reported 16.6 per cent by Farzan et al.The highest resistance was observed against the [16].Fadel et al.
investigation among 80 E. coli isolates from milk and Detection of virulence genes: In this study, out of 80 E.

Antibiotic sensitivity test:
Table-1).Standardization regard as an E. coli.Various biochemical tests such as of PCR was done by using standard strain of E. coli catalase test, Indole production, Methyl red, Voges O157:H7 and EPEC.The reactions were performed in proskauer, Simon's citrate agar, Urease production, the thermal cycler (Applied Biosystem, Sweden) with Nitrate reduction etc. were done for the confirmation of o pre-heated lid (Lid temp.105 C).For the confirmation E. coli as proposed by Edward and Ewing [9]. of targeted PCR amplification, consisting of 1 µl of 6X

Table - 1
. Details of primers used for PCR.

Table - 3
[16]mikacin are commonly used aminoglycosides in the Nanu et al.[17]and 33.96 per cent by Mohd et al. [18].All the E. coli isolates were found to be sensitive Prevalence of E. coli reported 28 per cent in cheese, the to Co-trimoxazole in the present study.But least reistance were recorded by Sabry and Elmalt [24], similar result 29.2 per cent in Ras cheese was recorded Onono et al. [25], Thaker et al. [22].Mohd et al. [18] by Fadel et al. [19].Higher prevalence 96 per cent in reported 60 per cent sensitivity to co-trimoxazole.cheesewasreportedbyPanetoetal. (2007).However,In the present study, 6.52 per cent of E. coli lower prevalence of E. coli (12.9%) in cottage cheese isolates from raw milk and various milk products were was reported by Singh and Prakash[20]and 16.6 per resistant to Cefotaxime, in contrast with present cent in cheese was reported by Farzan et al.[16].findinghigher resistance was recorded by Mahami et Prevalence of ice-cream was found 20 per cent in

Table - 3
. PCR amplification of virulence genes