Bacterial Identification By Ribotyping Technique
In prokaryotic cells, the rrn ribosomal operon is made up of three subunits which encode for the 16S, 23S and 5S rRNA genes. These genes are linked together by internal transcribed spacer (ITS) regions which contain tRNA’s and are controlled by a single promoter and terminator. Located between these rRNA encoding regions are intergenic spacer regions, which vary in size and sequence. The number of rrn ribosomal operons within a bacterial genome varies between different bacterial species, some bacteria such as Mycoplasma have just one copy of the rrn operon while E. coli has 7 copies of the rrn operon within its genome. The differences between the spacer region of the rrn operons among bacterial species/strains are used in phylogenetic studies as the polymorphisms can be detected by using techniques such as polymerase chain reaction (PCR) ribotyping followed by agarose gel electrophoresis.
PCR is a ribotyping technique used in the analysis of the different variable regions within the rrn operon, where the spacer regions are amplified using primers followed by digestion using specific restriction enzymes. ITS-PCR is a type of PCR technique which specifically amplifies the region between the 16S and 23S rDNA genes of the rrn operon, this was the technique used in this experiment. The amplicons generated by the ITS-PCR reaction are then ran on an agarose gel in order to separate them into bands based on their relative size. This allows for the visualization of the ITS-PCR amplicons as they appear as a profile of bands of different sizes on the produced electropherogram. Each of the bands in the produced profile for the sample being analyzed corresponds to the various ITS regions of the rrn operon. This in turn allows for the intraspecific/interspecific differentiation of bacterial strains/species based on their band profile by assigning each isolate to a genotype group using the number and sizes of the produced bands for each isolate on the electropherogram.
Materials & Method
The experiment was carried out as outlined in the Laboratory Skills Manual pg. 40-47. The exact method carried out and any deviations which were made to the protocol were recorded in the laboratory notebook.
Results
The bacterial species which was examined by this group was Serratia marcescens, isolates of 3 strains of this bacterial species were examined (Sm1, Sm2, Sm3). A negative control which contained water instead of bacterial DNA was also prepared.
Discussion
From the obtained results visible on the electropherogram as can be seen above, it can be said that samples Sm1 and Sm2 are possibly the same strain as they both produced two strong bands in the same region of the MW ladder on the gel. The banding pattern obtained for the Sm3 isolate differed as the bands which were produced on the gel were located higher on the gel and hence it was assigned to a different genotype (B) as can be seen in Table 2 above. Sample Sm3 produced two strong bands and also two very faint bands which can be seen above the two main bands, these bands were of higher MW.
Similar banding patterns were observed for the Klebsiella pneumonia and Staphylococcus aureus isolates as samples 1 and 2 for both of them were assigned to genotype A while samples 3 of both were assigned to genotype B. Although the banding pattern for Serratia marcescens, Klebsiella pneumonia and Staphylococcus aureus was similar in a way that resulted in the same genotype groups to be assigned to the same isolate numbers, the ITS-PCR profiles of the three-different species varied proving that this method is suitable for interspecies differentiation.
For the Sm3 Escherichia coli and the Klebsiella oxytoca isolates no banding pattern could be observed on the electropherogram hence it could not have been assigned to a genotype group. The lack of bands could perhaps indicate that the PCR did not work or there was no bacterial DNA contained in the PCR sample, the sample also could have not been loaded into the well properly. On the electropherogram for the Escherichia coli isolates there was no MW ladder visible so there clearly was a problem with loading of the samples onto the wells in the gel or the staining did not work. For the Klebsiella oxytoca isolates it can also be observed that there is still some of the sample close to the well which could mean that the sample molecules were too big for the % agarose use so if the gel % was decreased making the pores in the matrix larger thus allowing the sample to travel through the gel.
All of the gels could have been ran for a little or at a higher voltage to maximize band separation. Also, further analysis of the bacterial strain could be performed in order to fully identify and characterize the types of strains of the analyzed bacterial species.
In conclusion
As the ITS-PCR profiles differed for the Serratia marcescens isolates it can be concluded that this method is suitable for intraspecies differentiation. As the ITS-PCR profiles for all of the analyzed bacterial species differed from one another it can also be concluded that this method is suitable for interspecies differentiation.
