Received for publication,
Department of Microbiology,
Nagercoi – 629 501, Kanyakumari
for Marine Science and Technology,
Rajakkamangalam- 629 502,
*Corresponding author: email@example.com; Ph:+91-4652-253078
Maintenance of the microbial quality and
safety of water systems is imperative as their fecal contamination may exert
high risks to human health as well as result in significant economic loss.
Human fecal material is generally perceived as constituting a greater human
risk than animal fecal material, considering that it is livelier to contain human
specific enteric pathogens . Determining the source of fecal pollution
enables as to apply appropriate management plans to remedy the problem and to
prevent any further contamination. E. coli
is now recognized as an important human pathogen of public health concern .
Infection with E. coli strains,
especially of 01 57:47, which may not apparent, results in a spectrum of
disease ranging from mild, non-blood diarrhoea to hemolytic
– Uraemic syndrome. Being considered as a ubiquitous organism widely
distributed in the environment, E. coli
deserves in depth investigation. In the
present study, the assessment of Randomly Amplified Polymorphic DNA (RAPD)
analysis was performed through the genotypic
source tracking method to identify the genetic variation of E.coli isolates derived from five
different sampling stations of a minor estuarine environment in the southwest
Materials and Methods
The present study was undertaken in four
different sampling stations of Rajakkamangalam estuary, a minor estuary in the Southwest
The E. coli cell pellets were ground individually in a glass homogenizer with 300ml of CTAB (cetyltrimethyl ammonium bromide) DNA extraction buffer (1% W/V CTAB; 1.4M NaCl; 10mM EDTA (pH 8.0); 100m Tris-HCl (pH 8.0); 0.2% V/V meracaptoethanol). The individual mixture was emulsified with equal volume of phenol: chloroform (1:1) and centrifuged at 10,000 X g for 5min at room temperature. The aqueous phase was collected and mixed with equal volume of chloroform: isoamyl alcohol (24:1). The mixture was then centrifuged at 10,000 X g for 5min and the ethanol was air-dried. The pellet was dissolved in 50ml of TE buffer (Tris 10mM- pH 8.0 and EDTA 1mM- pH8.0).The isolated DNA of the strains was quantified by spectrophotometer (260nm) and quality was tested by agarose gel electrophoresis.
The DNA (20mg) was dissolved in 20ml PCR reaction
buffer containing 10mM Tris-HCl (pH 9.0), 1.5mM MgCl2, 50mM KCl,
0.01% gelatin, 0.2mM dNTPs, 21 pM of primer and 0.5U of DNA polymerase. Ten
primers (RAPD Kit A1 to A10) obtained from IDT were used for RAPD - PCR
studies. PCR was conducted according to the method described by Williams et al. : initial heat step (94oC
for 5 min.), 40 cycles of denaturation (94oC for 1 min.), annealing
(36oC for 1min.) and extension (72oC for 2min.) and a
finial extension step (72oC for 7 min.). Amplification was performed
using a programmable thermal Cycler PTC-150 (MJ Research,
Sequence of the Primers:
RAPD Kit A1 5’CAGGCCCTTC3’
RAPD Kit A2 5’TGCCGAGCTG3’
RAPD Kit A3 5’AGTCAGCCAC3’
RAPD Kit A4 5’AATCGGGCTG3’
RAPD Kit A5 5’AGGGGTCTTG3’
RAPD Kit A6 5’GGTCCCTGAC3’
RAPD Kit A7 5’GAAACGGGTG3’
RAPD Kit A8 5’GTGACGTAGG3’
RAPD Kit A9 5’GGGTAACGCC3’
RAPD Kit A10 5’ GTGATCGCAG3’
Molecular markers offer many options to differentiate intra and inter specific populations. Molecular identification of microbes has been frequently conducted on the basis of existence of demonstrative polymorphic DNA fragments amplified by PCR . It has been suggested that the outcome of a RAPD reaction was in part determined by a competition for priming sites in the Genome . Amplification is probably initiated at many sites, but only a subset of all possible products is detected as visible bands after amplification [7, 8]. In the present study, the RAPD analysis was performed for five E. coli isolates with RAPD kit A10 for their ability to differentiate intra species variation. The primer RAPD kit A10 amplified two fragments of 1480 and 1870 base pairs (bp) in E. coli isolate 1. E. coli isolate 2 amplified single fragment of 1480 bp with magnified size. The RAPD product of 1870 bp fragment was present in E.coli isolate 3. In E. coli strain 4, the RAPD product 3900 bp to 913 bp were amplified with low intensity. The E. coli strain 5 amplified five fragments with base pair ranged from 3090 to 1200 bp. For comparison of molecular variation a 6th isolate namely Pseudomonas Sp. was also simultaneously analyzed with RAPD kit A10. In this isolate five fragments with base pair ranging from3530 to 831 bp were amplified (Fig.)
The RAPD profiles of E. coli strains and also Pseudomonas sp. obtaining the present study were used for the calculation of similarity index. The RAPD fragments of E. coli strain 1 has the similarity index of 0.29 with isolate 2 and 0.22 with isolate 4.
In E. coli strain 2, the RAPD fragments had the similarity index of 0.20 with isolate 4. The E. coli strain 3 had the similarity index of 0.22 and 0.25 with E. coli strain 4 and 5 respectively. The E. coli strain 5 had the similarity index of 0.18 with isolate 4. However the isolate 6 (Pseudomonas Sp) has no similarity index with the RAPD fragment of any of the other tested strains.
Dendograms in the phylogram form depicting the phylogenetic relatedness between various isolates and Nels’ genetic homology coefficients generated by the unweighed pair group method using arithmetic averages (UPGMA) is presented in Figure. Examination of dendogram indicated that the selected E. coli isolates were grouped into three major clusters excluding the Pseudomonas sp, which formed a separate cluster. E. coli isolates 1 and 2 with 29% homology coefficient were clustered together as the same species. With 25% homology co-efficient of E. coli strains 3 and 5 were clustered together as the same species. Isolate 4 was grouped with a third cluster forming solitary cluster with isolate 1 and 2.
The variations noted in the Nel’s genetic homology co-efficient of E. coli isolates in the present study may be attributed to environmental conditions prevailed in the sampling sites. Though the E. coli isolates 1 and 2 were obtained from two different stations, due to environmental similarity in those stations, these isolates were clustered together as the same species. E. coli isolates 3 and 5 were clustered together as the same species because these strains were isolated from the same station and they also indicated from the influence of unlike environment variables in that station. E. coli strain 4 isolated from 4th station was groups as a separate cluster also indicated the different environmental variables influence on the genetic homology coefficient. Test results showed the influence of variation in environmental variables on genetic variation of E. coli strains isolated from the estuarine environment.
Our findings are consistent with Naveen Kumar et al. , who isolated 5 strains of septicemia and diarrheic E.coli 078. from sheep and calves and reported that there was much polymorphism between isolates and the sheep isolates were more similar with each other than isolates from calves by RAPD method with seven random Primers viz. OPB1, OPB2, OPB5, OPB6, OBP7, OBP8 and OPB9. They further inferred that, high homogeneity among isolates from sheep and lower homogeneity among isolates from cleave may probably be due to the fact that the former isolates were cultured from the same farm, having endemic infection; whereas, the later isolates were cultured from different locations though with similar disease condition. Akopyanz et al.  also reported that, the 60 strains of Helicobacter pylori isolated from the patients at one hospital were found to be distinguishable from each other by RAPD methods with each of the two arbitrary primers tested.
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