Study on occurrence and antibiogram pattern of Escherichia coli from raw milk samples in Anand, Gujarat, India

Aim: The study was carried out with aim to isolate Escherichia coli from raw milk samples and determine antibiogram pattern of E. coli isolates. Materials and Methods: During 6 months duration of study a total of 100 raw milk samples were collected from different places in and around Anand city such as individual household, cattle farms, milk collection centres of Co-operative milk dairies and milk vendors. All raw milk samples were enriched in peptone water and inoculated on selective media and various biochemical tests were performed for confirmation of isolates. Antibiogram pattern of E.coli to antimicrobial agents was evaluated by disk diffusion method. E. coli isolates were sent to National Salmonella and Escherichia Centre, Central Research Institute, Kasauli, Himachal Pradesh for serotyping. Results: The result of present study revealed that out of 100 samples, 38 samples were found contaminated with E.coli. Antibiogram pattern revealed high resistance against ampicillin (100 %), whereas moderate resistance was observed for streptomycin (57.89 %), oxytetracycline (47.37 %) and amoxy-clav (42.11 %). Also lesser percentage of resistance was observed for co-trimoxazole (13.16 %) and chloramphenicol (5.26 %). Serotypes detected were O24 (7 isolates), followed by


Introduction
microorganisms which constitute a public health hazard [11].E. coli is one of the main inhabitants of the Food safety and quality is a topic of public intestinal tract of most mammalian species, including concern throughout the world.Well publicized and humans and birds.Most E. coli are harmless, but some widespread food borne disease outbreaks have created are known to be pathogenic bacteria, causing severe an awareness of potential threats to human health from intestinal and extra intestinal diseases in man [12].The food products.Markets and consumers for raw milk milk sold in raw forms and because of possibilities of and their products have existed in many parts of the contamination with E. coli posses a great hazard to world.Raw or processed milk is a well-known good public health [13].medium that supports the growth of several microbes In addition to the presence of E. coli denoting with resultant spoilage of the product or infections/ fecal contamination, the presence of virulenceintoxications in consumers [1, 2].related genes in E. coli strains refer to the patho-Generally, bacteria in the milk can occur through genicity of the isolates.Previous studies documented colonization of the teat canal or an infected udder the presence of some E. coli isolates from raw milk and (clinical and subclinical mastitis) or gets contaminated products for virulence markers [14][15][16][17].at various stages be it from the animal, milker (manual Thus, the objective of this study was to investigate as well as automated), extraneous dirt or unclean water the occurrence of the opportunistic pathogen E. coli in [3 -7].Many microorganisms can get access to milk raw milk, determine antibiogram pattern and serotyping and milk products, among these are E. coli.Coliforms of E. coli isolates.and E. coli are often used as marker organisms [8,9,10].Recovery and counting of E. coli is used as Materials and Methods

reliable indicator of fecal contamination and indicates
Sample collection: From February 2011 to July a possible presence of enteropathogenic and/or toxigenic 2011, a total of 100 raw milk samples were randomly isolates to different antimicrobial agents was done by collected in sterilized screw capped bottles from disk diffusion method using commercial disks [19] different localities in and around Anand city such as procured from HiMedia Pvt. Ltd. and almost all individual household, cattle farms, milk collection antimicrobial agents were having expiry date after 5-6 centres of Co-operative milk dairies and milk vendors.months of completion of research.
Serotyping of E. coli isolates: Isolates were sent to National Salmonella and Escherichia Centre, Central

Isolation and identification of Escherichia coli:
Isolation of E.coli was attempted according to Singh Research Institute, Kasauli, Himachal Pradesh.and Prakash [18] with slight modification.A part of

Results
each sample (10 ml) was enriched in peptone water (HiMedia Pvt.Ltd.) (90 ml) and was incubated at 37 In the present study, it was revealed that 38 °C for 24 hours.Enriched samples were inoculated on samples (38.00%) out of 100 raw milk samples were Mac Conkey Agar (MCA) (HiMedia Pvt.Ltd.) a dual found positive for Escherichia coli according to purpose (selective and differential) medium, by four morphological and cultural characteristics (Table 1) flame technique and plates were incubated at 37 °C and biochemical tests (Table 2).for 24 hours.Pink coloured colonies appeared were The antibiogram pattern revealed that 38 E.coli considered as presumptive of E.coli.

Discussion
Antibiogram pattern of the isolated E.coli to some antimicrobial agents: The susceptibility of E.coli is not only regarded as an indicator of  fecal contamination but more likely as an indicator of indicator organism not only indicates the poor hygiene poor hygiene and sanitary practices during milking but also itself may be pathogenic.The pathogenic and further handling.Higher prevalence of E.coli was bacteria such as E.coli may pass to the milk; this reported by Ali and Abdelgadir [11] who found 63 % suggests that raw milk should be considered a vehicle prevalence and Lingathurai and Vellathurai [20] who for the transmission of potentially pathogenic bacteria.found 70 % prevalence.The incidence of the species of For this, hygienic milk production techniques should E. coli itself in milk and milk products, as a possible be followed such as regular sterilization of dairy cause of food born disease, is not significant if E. coli equipment, washing of utensils, milker's hands, udder, is normally an ubiquitous organism [21], yet the , etc.As well as pasteurization or boiling of milk prior pathogenic strains if present could be harmful to to consumption is required.Frequent use of antibiotics consumers.should be stopped as antibiotic resistant strains are Coliform bacteria can be carried into milk duct continuously increasing.This study highlights the of the cow during milking by suction of the milking need for continuous surveillance of antibiotic machine and then flushed out during subsequent sensitivity pattern of E.coli with a view to selecting milking without causing clinical symptoms of appropriate therapy.infection.Previous studies provided evidence that and O128 have been found to be associated with distributors was unsafe.The presence of fecal

Table - 1
. Morphological and culture characteristics of bacteria

Table - 2
. Biochemical characterisation of E. coli Beutin et al. [31]and Nishikawa et al.[32].Thus, these ment food safety and public health implications.serotypesmaybe of zoonotic importance.In present Foodborne Pathogens and Dis., 2: 1115-1129.study,none of the above serotypes were found but still 3. Banwart, G.J.(1989).Microorganisms associated the virulence of E. coli should not be considered less.