ISOLATION AND ISOLATION AND CHARACTERIZATION OF CHARACTERIZATION OF E. E. coli coli FROM THE MEAT FROM THE MEAT SAMPLESSAMPLES ByBy

BHAWNA SINGHBHAWNA SINGH

Under the supervision ofUnder the supervision of Dr. PurushottamDr. Purushottam

, Sardar Vallabh Bhai Patel , Sardar Vallabh Bhai Patel University of Agriculture & University of Agriculture & Technology, Technology, Modipuram Modipuram

INTRODUCTION :INTRODUCTION :

Meat, a rich source of protein.Meat, a rich source of protein. Today the people of all religions eat the meat Today the people of all religions eat the meat

without thinking about the ethical values. without thinking about the ethical values.

Demerits:Demerits: Raw meat available in open-air local retail Raw meat available in open-air local retail

shops without appropriate temperature control. shops without appropriate temperature control. Changes in eating habits, mass catering, Changes in eating habits, mass catering,

complex and lengthy food supply procedures complex and lengthy food supply procedures with increased international movement and with increased international movement and poor hygiene practices. poor hygiene practices.

During food During food processing and processing and handling.handling.

Reason behind Reason behind choosing choosing E. coliE. coli as as a research material a research material is that its a is that its a fastidious bacteria fastidious bacteria which can grow which can grow easily in appropriate easily in appropriate laboratory laboratory condition. condition.

Objective of this study

To detect the presence of virulent genes in the meat sample

specifically in the Meerut zone.

Importance of this study:Importance of this study:To aware the people what is good for To aware the people what is good for

the health.the health.To know about bacterial To know about bacterial

contamination in the meat samples contamination in the meat samples specifically in Meerut zone.specifically in Meerut zone.

To detect the virulent genes if they To detect the virulent genes if they were present in the meat samples. were present in the meat samples.

To know Antibiotic profile that will To know Antibiotic profile that will control the use of antibiotics in control the use of antibiotics in clinical practice.clinical practice.

Procedure Procedure

Morphological characterization

Biochemicalcharacterization

Molecular characterization

Sample collectionSample collection

Table : Source & distribution of Meat sample S. NoS. No

SampleSample Place of collectionPlace of collection No. of sampleNo. of sample

AA MeatMeat JaidifarmJaidifarm 11

BB MeatMeat KutiKuti 11

CC MeatMeat PVS roadPVS road 11

DD MeatMeat TejgarhiTejgarhi 11

EE MeatMeat Hapur roadHapur road 11

FF MeatMeat GolakuanGolakuan 11

GG MeatMeat JiadinagarJiadinagar 11

HH MeatMeat Nai sarakNai sarak 11

II MeatMeat Shastri NagarShastri Nagar 11

JJ MeatMeat Bhumia pul shopsBhumia pul shops 11

KK MeatMeat Cantt areaCantt area 11

LL MeatMeat Pallavpuram Pallavpuram 11

MM MeatMeat Garh roadGarh road 11

NN MeatMeat Hind shopHind shop 11

O O MeatMeat Begum pul Begum pul 11

PP MeatMeat Abulane Abulane 11

QQ MeatMeat Jali coti Jali coti 11

RR MeatMeat Hapur haddaHapur hadda 11

S S Meat Meat Saket Saket 11

T T Meat Meat Budhana gateBudhana gate 11

Enriched in peptone solution for 24 hrs Enriched in peptone solution for 24 hrs at 37˚C.at 37˚C.

Streaked on Mac Conkey agar and Streaked on Mac Conkey agar and incubated at 37˚C for 24 hrs.incubated at 37˚C for 24 hrs.

Pink colonies were streaked on EMB Pink colonies were streaked on EMB agar and incubated at 37˚C for 24 hrs.agar and incubated at 37˚C for 24 hrs.

Metallic green sheen colonies were Metallic green sheen colonies were observed.observed.

Metallic sheen colonies streaked and Metallic sheen colonies streaked and stabbed on the Tsi slant and incubated stabbed on the Tsi slant and incubated overnight.overnight.

Isolation of Isolation of E. coli E. coli on on differential media:differential media:Meat sample Meat sample A A showing pink colour colonies on Mac Conkey agar.showing pink colour colonies on Mac Conkey agar.

Control ResultControl Result

Table: Meat samples Table: Meat samples A, B, E, J, R, TA, B, E, J, R, T showing showing positive test on Mac Conkey agarpositive test on Mac Conkey agar

Sample.No. Date of streak on MacConkey Agar

Incubation Period

Pink colourColonies

A 25-2-2011 24 hrs +

B 25-2-2011 24 hrs +

C 25-2-2011 24 hrs -

D 25-2-2011 24 hrs -

E 25-2-2011 24 hrs +

F 1-3- 2011 24 hrs -

G 1-3- 2011 24 hrs -

H 1-3 -2011 24 hrs -

I 1-3- 2011 24 hrs -

J 1-3- 2011 24 hrs +

K 5-3- 2011 24 hrs +

L 5-3- 2011 24 hrs -

M 5-3- 2011 24 hrs -

N 5-3- 2011 24 hrs -

O 5-3- 2011 24 hrs -

P 10-3-2011 24 hrs +

Q 10-3-2011 24 hrs +

R 10-3-2011 24 hrs +

S 10-3-2011 24 hrs -

T 10-3-2011 24 hrs +

Isolation of Isolation of E. coliE. coli on EMB on EMB agar:agar:

Meat sample A showing metallic green sheen colonies on EMB Meat sample A showing metallic green sheen colonies on EMB agar.agar.

Control ResultControl Result

Table: Meat sample A, B, J, K, P, R showing Table: Meat sample A, B, J, K, P, R showing positive test on EMB agarpositive test on EMB agar

S.No.S.No. Date of streak on EMB agar Date of streak on EMB agar

Incubation PeriodIncubation Period Green Sheen ColoniesGreen Sheen Colonies

AA 26-2-201126-2-2011 24 hrs24 hrs ++

BB 26-2-201126-2-2011 24 hrs24 hrs ++

CC 26-2-201126-2-2011 24 hrs24 hrs --

DD 26-2-201126-2-2011 24 hrs24 hrs --

EE 26-2-2011 26-2-2011 24 hrs24 hrs --

FF 2-3-20112-3-2011 24 hrs24 hrs - -

GG 2-3-20112-3-2011 24 hrs24 hrs --

HH 2-3-20112-3-2011 24 hrs24 hrs - -

II 2-3-20112-3-2011 24 hrs24 hrs --

JJ 2-3-20112-3-2011 24 hrs24 hrs ++

KK 6-3-20116-3-2011 24 hrs24 hrs + +

LL 6-3-20116-3-2011 24 hrs24 hrs --

MM 6-3-20116-3-2011 24 hrs24 hrs --

NN 6-3-20116-3-2011 24 hrs24 hrs - -

O O 6-3-20116-3-2011 24 hrs24 hrs - -

P P 11-3-201111-3-2011 24 hrs24 hrs ++

QQ 11-3-201111-3-2011 24 hrs24 hrs --

R R 11-3-201111-3-2011 24 hrs24 hrs ++

SS 11-3-201111-3-2011 24 hrs24 hrs --

TT 11-3-201111-3-2011 24 hrs24 hrs --

Morphological Morphological CharacterizationCharacterization

Gram staining:Gram staining: Gram staining of Gram staining of

isolate A.isolate A.

Biochemical characterization Biochemical characterization Antibiotic profile test:Antibiotic profile test:

Antibiotic and Antibiotic and symbolsymbol

Results Results

Ciprofloxacin [ Cf ]Ciprofloxacin [ Cf ] ResistantResistant

Co-Trimazine [Cm]Co-Trimazine [Cm] Resistant Resistant

Kanamycin [ K ] Kanamycin [ K ] Resistant Resistant

Nitrofurantoin [Nf ]Nitrofurantoin [Nf ] Sensitive Sensitive

Steptomycin [ S ]Steptomycin [ S ] IntermediateIntermediate

Tetracyline [ T ]Tetracyline [ T ] ResistantResistant

Amikacin [ Ak ]Amikacin [ Ak ] Resistant Resistant

Carbenicillin [ CbCarbenicillin [ Cb ] ] Resistant Resistant

Triple sugar iron test:Triple sugar iron test:Test is positive for sample A, B, J, K, P, R. Test is positive for sample A, B, J, K, P, R. Control ResultControl Result

E. coliE. coli Identification kit: Identification kit: The isolates of sample A is positive for all The isolates of sample A is positive for all

biochemical and carbohydrate test except biochemical and carbohydrate test except citrate test.citrate test.

Table : Biochemical test results Table : Biochemical test results obtained by identification kit.obtained by identification kit.

S. NoS. No TESTTEST RESULTRESULT

AA Methyl red test Methyl red test ++

AA Voges ProskaeurVoges Proskaeur’’s test s test - -

A A Citrate utilization test Citrate utilization test --

A A Indole testIndole test ++

A A Glucorinidase testGlucorinidase test ++

AA Nitrate reduction testNitrate reduction test ++

AA ONPG testONPG test ++

A A Lysine decarboxylase testLysine decarboxylase test ++

AA Lactose utilization testLactose utilization test ++

AA Glucose utilization testGlucose utilization test ++

A A Sucrose utilization testSucrose utilization test ++

AA Sorbitol utilization testSorbitol utilization test ++

Molecular characterization of Molecular characterization of E. E. coli coli : PCR: PCR

PCR was performed to detect virulent PCR was performed to detect virulent genes of VTEC by using three different genes of VTEC by using three different primer specific to vt1, vt2 and eae gene primer specific to vt1, vt2 and eae gene according to Blanco according to Blanco et. alet. al., (1996) . Base ., (1996) . Base sequence and predicted sizes of amplified sequence and predicted sizes of amplified product for the specific oligonucleotide product for the specific oligonucleotide primers (Genei, Bangalore) used as shown primers (Genei, Bangalore) used as shown in following table.in following table.

Primers used in PCR for Primers used in PCR for amplification of specific fragments amplification of specific fragments

for virulent genes of VTEC :for virulent genes of VTEC : GENESGENES SEQUENCESEQUENCE PRODUCT SIZEPRODUCT SIZE

vt1 avt1 a CAGTTAATGTGGTGGCGAAGCAGTTAATGTGGTGGCGAAG 894 894

vt1 bvt1 b CTGCTAATAGTTCTGCGCATCCTGCTAATAGTTCTGCGCATC 894894

vt2 avt2 a CTTCGGTATCCTATTCCCGGCTTCGGTATCCTATTCCCGG 478 478

vt2 bvt2 b GGATGCATCTCTGGTCATTGGGATGCATCTCTGGTCATTG 478478

eae 1eae 1 ACGTTGCAGCATGGGTAACTCACGTTGCAGCATGGGTAACTC 815815

eae 2 eae 2 GATCGGCAACAGTTTCACCCG GATCGGCAACAGTTTCACCCG

815 815

Master mix preparation

Taq Buffer = 2.5µl x 6 =15.0µldNTPs = 2µl x 6 =12.0µlPrimer( i) = 2µl x 6 =12.0µlPrimer( ii) = 2µl x 6 =12.0µlTaq poly = 0.5µl x 6 =3.0µlDW = 11.0µl X 6 =66.0µl

Procedure

1ml sample( enriched in peptone solution ) in eppendorf and then boil for 10 min & left for some time on ice

20µl master mix +5µl sample in eppendorf

Pour in PCR tubes and kept on ice.

Run the PCR

Electrophoresis

No bands observed

ConclusionConclusion

The meat available to the consumer contain The meat available to the consumer contain a high a high E. coli E. coli contamination.contamination.

The samples taken from the area such as The samples taken from the area such as Jaidifarm, Kuti, Hapur Hadda are highly Jaidifarm, Kuti, Hapur Hadda are highly bacterial contaminated. bacterial contaminated.

The area such as PVS road, Abulane, Saket, The area such as PVS road, Abulane, Saket, are free from bacterial contamination. are free from bacterial contamination.

The samples are free from virulent genes The samples are free from virulent genes which is the major cause of pathogenicity which is the major cause of pathogenicity so its good for the people’s health.so its good for the people’s health.

THANK YOUTHANK YOU