Available at www.veterinaryworld.org/Vol.6/Sept-2013/15.pdf RESEARCH ARTICLE Open Access Isolation of pathogenic Escherichia coli from stool samples of diarrhoeal patients with history of raw milk consumption

Aim: To detect the occurrence of pathogenic Escherichia coli from stool samples of diarrhoeal patients with history of raw milk consumption and to determine the public health significance of isolates, especially their role in causing human diseases. Materials and Methods: A total of 100 stool samples from diarrhoeal patients, with history of raw milk consumption were collected from primary health centres in and around Anand city, under aseptic conditions and a total of 50 raw milk samples were collected from milk vendors, retail shops located in Anand city in sterilized sample bottles. MacConkey broth was used for the enrichment of all the samples and inoculation was done on MacConkey agar and EMB agar was used as the selective media. This was followed by the confirmation of isolates using biochemical tests. For the serotyping, E. coli isolates were sent to the National Salmonella and Escherichia Centre, Central Research Institute (CRI), Kasauli, Himachal Pradesh. Detection of virulence genes was performed using PCR technique. Results: During the present investigation, 26 (52%) E. coli isolates from 50 milk samples and 59 (59%) E. coli isolates from 100 stool samples were recovered. Out of 85 E. coli isolates sent for serotyping, 74 isolates could be typed which were further distributed into 13 different serogroups O2, O4, O8, O17, O22, O25, O29, O36, O45, O60, O90, O116 and O172, whereas 8 isolates were found untypable and 3 isolates were reported rough isolates. Of the 59 E. coli isolates from stool samples of diarrhoeal patients tested, 15 isolates (25.42%) were reported to be positive for stx genes, among that 6 (10.16%) were positive for stx1 gene, 9 (15.25%) isolates were positive for stx2 gene, while 3 isolates (5.08%) were positive for eaeA gene. In this study, 21 E. coli isolates were found to be Shiga toxin producing E. coli (STEC) while none of the isolates were positive for the serotype O157. Conclusions: Our present findings indicate that raw milk may act as a source of pathogenic E. coli and it may be responsible for the occurrence of diarrhoea and various other health-related complications in humans. We therefore recommend proper managemental practices and effective control measures for improved hygiene and sanitation.


Introduction
organism, and they consist of two types of toxins; stx 1 and stx .STEC may be responsible for various disease 2 Escherichia coli is a facultative anaerobe, Gram outbreaks across the world.In Italy, an outbreak of E. negative and belongs to the family Enterobacteriaceae, coli O26 was reported during 2005, which occurred and is usually a commensal organism [1].Enteroupon consumption of buffalo milk products [5].The pathogenic E. coli which affect humans are categorised outbreak of haemolytic uremic syndrome was caused into six groups: Shiga toxin producing E. coli (STEC), by O145:H28 and O26:H11 which was associated with enteropathogenic E. coli (EPEC); entero toxigenic E.
undertaken to detect the occurrence of pathogenic E. STEC is a group of food borne pathogenic coli from stool samples of diarrhoeal patients with microorganisms linked to a broad range of human history of raw milk consumption but not from those diseases, ranging from diarrhoea to hemorrhagic colitis who consumed boiled milk, collected from primary (HC), thrombocytopenia, hemolytic uremic syndrome health centres in and around Anand city.(HUS), and can also lead to human mortality [3].STEC represents the only pathogenic group of E. coli having a

Materials and Methods
zoonotic significance [4].Shiga toxins are the major Sample collection: From September 2012 to February virulence factors contributing to pathogenicity of the 2013, a total of 50 raw milk samples (50 ml) were collected from milk vendors, retail shops located in Anand city in sterilized sample bottles and a total of 100 stool samples from diarrhoeal patients with history of raw milk or milk product consumption were a thermal cycler (Applied Biosystems, Sweden) with collected from primary health centres in and around pre-heated lid (Lid temp.105ºC).For the confirmation Anand city.Samples from patients were collected of targeted PCR amplification, 1 µl of 6X gel loading preferably before the initiation of antimicrobial buffer along with 5 µl of the PCR product was therapy using multipurpose sample collecting bottles electrophoresed along with DNA molecular weight and were brought to the Post Graduate Research marker (Gene Ruler, MBI Fermentas).Agarose gel Laboratory of the Veterinary Public Health department (2%) along with ethidium bromide (at the rate of 0.5 in an ice box for further processing and microbio-µg/ml) was used.sent for serotyping, 74 isolates could be typed which were distributed into 13 different serogroups, whereas Serotyping of E. coli isolates: E. coli isolates recovered 8 isolates were found untypable and 3 isolates were from milk and stool samples were serotyped based on reported to be rough (Table -2).Among the 42 isolates, their somatic (O) antigens at the National Salmonella the different serogroups detected in the descending and Escherchia Centre (NSEC), Central Research order were 16 isolates (18.82%) of O90; 12 isolates Institute (CRI), Kasauli, H. P., India.
(14.11%) each of O60 and O4; 9 isolates (10.58%) each DNA isolation: DNA was isolated from E. coli by using of O2 and O116; 7 isolates (8.23%) of O8; 3 isolates boiling method [9].Approximately a loopful of culture (3.52%) of O25; 1 isolate (1.17%) each of O17, O22, was mixed with 100 µl of sterilized DNAse and RNAse O29, O36, O45 and O172.free water in a micro centrifuge tube.This was followed Detection of virulence genes: In present study, out of by denaturation at 95ºC for 10 min using the thermal 85 E. coli isolates from milk and stool samples tested, cycler (Applied Biosystems, Sweden).Finally, cellular 21 isolates (24.70%) were reported to be positive for debris was removed by centrifugation (10000 rpm for 5 stx genes, among them 8 (9.41%) were positive for stx 1 min) and 3 µl of the supernatant was used as a DNA gene, 13 (15.29%)isolates were positive for stx gene, 2 template in PCR reaction mixture.while 4 isolates (4.70%) were positive for eaeA gene.

Polymerase chain reaction (PCR): Screening of all the
In this study, 21 E. coli isolates belong to STEC, while E. coli isolates was done to detect the presence of all the isolates were negative for serotype O157 (Table-3).virulence associated genes using the PCR technique.

Discussion
The PCR was standardized for the detection of stx , stx , 1 2 eaeA and rfbO157 following the methods described by Raw milk is a good source of various pathogenic Osman et al. [10] for detection of stx ,stx and eaeA microorganisms.Various pathogenic bacteria can grow genes and methods described by Dhanashee and and survive in the raw milk.A multitude of factors are Mallaya [11] for detection of rfbO157 with suitable responsible for the occurrence of E. coli in milk.

modifications (Table-1). Standardization of PCR was
Contamination of milk with E. coli may occur due to done using standard reference strain of E. coli unhygienic conditions, poor management, improper O157:H7 and EPEC.The reactions were performed in storage conditions and improper processing of milk.Unhygienic conditions during storage and supply of virulence genes reported from raw milk were also milk for human consumption may be responsible for recovered from stool samples which indicate that raw the occurrence of pathogenic E. coli in milk.
milk may be responsible for the occurrence of Consumption of contaminated milk by humans may virulence genes of E. coli in humans.The report of produce pathogenic conditions like diarrhoea, virulence genes suggest that prevalence of STEC in hemorrhagic colitis (HC), hemolytic uremic syndrome raw milk may be involved in outbreaks of human HC (HUS) etc. and HUS and is of significant importance from the In present study, of the 59 E. coli isolates from viewpoint of public health and hygiene.stool samples of diarrhoeal patients tested, 15 isolates Among bacterial infections E. coli has a (25.42%) were positive for stx genes, among them 6 significant role in causing severe food poisoning in (10.16%) were positive for stx gene, 9 (15.25%)man.Many diseases have appeared due to changes in 1 domestic animal managemental practices, food isolates were positive for stx gene, while 3 isolates 2 industry and changes in feeding habits of humans.
(5.08%) were positive for eaeA gene.During the The specific serogroup of E. coli can be linked to present investigation, the stool samples were collected the occurrence of certain clinical conditions.Hygienic from diarrhoeal patients with history of raw milk condition, geographical area and prevalence of E. coli consumption, and the occurrence of E. coli in stool in humans and animals in a particular region may be samples may be due to the consumption of raw milk responsible for the distribution of different serotypes of contaminated with E. coli.In the present study, the in future studies.

Prevalence of E. coli: During
the present investigation, MacConkey agar and incubated for 24 hrs at 37ºC.Pink 26 (52%) E. coli isolates from 50 milk samples and 59 coloured colonies were sub cultured on Eosin (59%) E. coli isolates from 100 stool samples from Methelene Blue (EMB) agar.Colonies producing diarrhoeal patients with history of milk consumption greenish metallic sheen on EMB agar were considered were recovered.as having E. coli.In addition, various biochemical tests were done for the confirmation of E. coli as proposed Serotyping of E. coli isolates: Out of 85 E. coli isolates by Edward and Ewing [8].

Table - 1
. Details of primers used for PCR reaction

coli isolates positive from Milk samples Stool samples
the zoonotic significance of these serotypes.21 E. 10.Osman, K. M., Mustafa, A. M., Elhariri, M. and coli isolates were found to be STEC in the present AbdElhamed, G. S. (2013) The Distribution of Escherichia investigation, which have significant role in causing Figure-1.Agarose gel showing PCR amplified product (614 bp) for stx1 gene in E. coli isolates.P: Positive control, N: Negative control, L: DNA Ladder, stx1: Positive samples Figure-2.Agarose gel showing PCR amplified product (779 bp) for stx2 gene in E. coli isolates.P: Positive control, N: Negative control, L: DNA Ladder, stx2: Positive sample Figure-3.Agarose gel showing PCR amplified product (384 bp) for eaeA gene in E. coli isolates.P: Positive control, N: Negative control, L: DNA Ladder, eaeA: and coli serovars, virulence genes, gene association and HC and HUS in humans.A number of untypable combinations and virulence genes encoding serotypes in pathogenic E. coli recovered from diarrhoeic calves, sheep isolates were also found in our study.We presume that and goat.Transbound Emerg.Dis.60(1):69-78.these isolates might belong to some rare or different 11.Dhanashree, B. and Mallya, P. S. (2008) Detection of shigaserotypes which are not yet identified from milk and toxigenic Escherichia coli (STEC) in diarrhoeagenic stool & milk products.They certainly deserve special attention meat samples in Mangalore, India.Indian J. Med.Res.128: 271-277.