ISOLATION AND CHARACTERIZATION OF E. coli FROM THE MEAT SAMPLES By By BHAWNA SINGH BHAWNA SINGH...
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Transcript of ISOLATION AND CHARACTERIZATION OF E. coli FROM THE MEAT SAMPLES By By BHAWNA SINGH BHAWNA SINGH...
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