Role of Two Different Annealing Temperatures in Genetic Variability Determination of Pseudomonas aeruginosa

Seven isolates of Pseudomonas aeruginosa were obtained from bacterial strain bank department of Biology/ College of Science/ Mosul University. The DNA was extracted from bacterial culture. Genetic variability was achieved by (RAPD-PCR), and ERIC2 primer was used. Two programs were used in thermal cycling containing two different annealing temperatures, the first was 50 o C, its amplification and electrophoresis results showed that all seven isolates had no band except the isolate No. (3) which has revealed a band (approximately 600 bp) when compared with DNA ladder, so this temperature was not suitable for detection of genetic variation for our local bacteria. The annealing temperature for the second program was 35 o C, its amplification and electrophoresis result has illustrated that there are genetic variability among just three isolates which had number 2, 3, and 4, while the other isolates have failed to show any band. So, the 35o C was better than 50o C to determine genetic variation in this study.


INTRODUCTION
Due to the plasticity of the phenotypic characteristics, molecular techniques (which are inherently more stable than phenotypic characteristics) have gained popularity for strain differentiation and epidemiological studies of many organisms (Deschaght et al., 2011).
Molecular typing of microbial pathogens is of pivotal importance in the elucidation of transmission routes, detailed genetic analysis at the species level gives insights into the variability within a bacterial population and generates evidence on genome plasticity and evolution, which in turn leads to bacterial adaptation to various environmental conditions. (Renders et al., 1996) For the last two decades, PCR-based genotyping methods have played an important role in bacterial typing schemes. One of the PCR-based methods, namely random amplification of polymorphic DNA (RAPD)-PCR, also known as arbitrarily primed-polymerase chain reaction (AP-PCR), has been described to be useful on account of its simplicity and utility for analysis of large throughput samples (Gürtlet and Mayall, 2001). Molecular research has been performed to study diversity among P. aeruginosa strains, polymorphism of certain of its genes and also genetic comparison of P. aeruginosa isolates from different hosts and environments (Onasanya et al., 2010 ;Al-Zahrani et al., 2012). The aim of the present study was to determine the genetic variation of seven clinical isolates of P. aeruginosa by random amplification of polymorphic DNA technique -using two annealing temperatures 50 o C and 35 o C which may give a significant look into the interrelationship of these bacterial isolates that in turn leads to investigate the risk of patient-to-patient transmission.

MATERIALS AND METHODS
Bacterial strains: Seven P. aeruginosa isolates were obtained from Bacterial strain bank/ Biology department/ college of science/ Mosul university) collected from the different clinical samples.
Colonies having the typical P. aeruginosa morphology were selected and placed in selective and differential media. Biochemical testing was performed to determine the bacterial genus and species, So the purity and identity of each isolate were confirmed by standard microbiological methods. (Forbes et al., 2007 ;Todar, 2011).

Preparation of Chromosomal DNA
The DNA was extracted from bacterial culture of P. aeruginosa using the method of phenolchloroform procedure (Ausubel et al., 2002). DNAs were stored at -20 o C until use. Baterial DNA Amplification: 1-The Primer: The RAPD primer ERIC2 (enterobacterial repetitive intergenic consensus) sequence (5' -AAG TAA GTG ACT GGG GTG AGC G -3') which is considered as universal primer has been used according to (Tazumi et al., 2009;Van daele et al., 2006;Speijer et al., 1999 ;Renders et al., 1996). The primer was supplied by BIONEER company as a lyophilized product of picomoles concentration, BIONEER company protocol was adopted for primer resuspension to bring as a final concentration to 5 picomoles/µl with nuclease-free water.

2-Reaction solution: Twenty five µl PCR reaction mix contained the ingredients listed in Table
(1)  At the end of the first amplification,(10 μl) were removed from each reaction mixture and examined by electrophoresis (100 V, 90 min) (ENDURO TM electrophoresis system, ENDURO power supplies, Labnet international. Inc. model: E 0303, Taiwan.) in gels composed of 2% (w/v) agarose stained with ethidium bromide (5 μg/100 ml) by using TAE buffer (40 mM Tris, 20mM acetic acid, 1mM EDTA, pH 8.3), Stained agarose gel was visualised under UV illumination by (UV transilluminator,Taiwan). A photograph was taken by using digital camera (Canon, Japan). The molecular weights of bands were estimated by using standard molecular markers (100 bp DNA Ladder, BIONEER).
The second amplification was changed just in annealing temperature of 35 o C (Table 3)   Then the procedure was completed as previous steps and the reaction mixture was completed as explained in the first program.

RESULTS AND DISCUSSION
After the extraction of genomic DNA from the seven P. aeruginosa isolates, it was amplified by RAPD-PCR using ERIC2 primer, then the products were separated by electrophoresis to study genetic variation in these local bacterial isolates.
The results of the first program did not give successful finding as seen in (Fig. 1); All seven isolates did not appear any band except the isolate No. (3) which has showed a band with molecular weight of approximately 600 bp compared with 100 bp DNA ladder (lane 3 in Fig. 1), so the isolate number (3) was different from the other six isolates by this band only. The absence of bands for other six isolates might be attributed to lack of complementary sequences to the primer in this temperature among the genomic DNA from P.aeruginosa (Devos and Gale, 1992).
The present study concluded that this temperature is not the optimum for determination the genetic variation of our bacterial isolates.
Although it has been reported by various groups that RAPD typing is reproducible, historically there have been several reports of problems associated with its reproducibility. Grif et al. (1998) reported that RAPD suffered from the lack of standardization using different methods in different laboratories and this may affect the quality of results. RAPD-PCR can also suffer from the same factors that affect ordinary PCR, including magnesium concentration, primer and reagent 'batch-to-batch' variation, annaeling temperature. and quality of the thermal cycler. Power (1996) suggested that for RAPD to be a definitive typing technique, the reproducible generation and interpretation of RAPD fingerprints needs to be developed, to achieve this criterion, the use of an automated system for DNA preparation combined with the use of manufactured RAPD master mixes, as well as use of the same thermal cycler and standard (Jamasbi and Proudfoot, 2008).
According to a previous result, annealing temperature was decreased to 35 o C according to the researchers (Van daele et al., 2006;Van daele et al., 2005).
In attempt to detect on distinct genetic variations among the isolates, 35º C was shown to be better than 50º C in demonstrating the genetic variations among the isolates.
Variations among studied isolates and RAPD results analysis are achieved by depending on presence or absence of amplified bands, also by the difference of molecular weights for these bands which differ by different sites number complemented to primer sequence on genomic DNA, and also differ by the distance between site and another (Mayer et al., 1997).
Our PCR products data indicated high diversity of P. aeruginosa, by using the primer ERIC2, this study showed the presence of genetic variation in just three isolates among P.aeruginosa as can be seen in (Fig. 2). Results analysis for polymorphism is usually scored in three different forms in experiments: presence or absence of amplified DNA bands, differences in molecular weight of amplified DNA bands and differences in the intensity of amplified bands (Bardakci, 2001).
The isolate No.4 revealed a largest numbers of amplified bands (6 bands) represented by <100, 300, 365, 410, 800 and 1000 bp. while the isolate No. 2 had four amplified bands represented by <100, 300, 400 and 600 bp, but three bands (100, 400, 650 bp) for the isolates No.3. ( Table 4). This indicates that the primer has found specific areas that bind it along with the DNA template, consequently amplification of the fragments was achieved, bands have appeared, and finally polymorphism has demonstrated. The other isolates of P. aeruginosa (which had number 1, 5, 6, and 7) had no revealed bands. This technique utilizes a variable short length arbitrary primer, and is advantageous, as it does not require any previous knowledge of the target DNA sequence data. The primer is amplified arbitrarily at low stringency, where the oligonucleotide binds at complementary and partially mismatched sites and generates bands which differ in length and nucleotide composition. (Tazumi et al., 2009).
As the primer efficiency depends on the total number of bands amplified by the primer and this could include a number of common ones (representing conserved) sequences among various species, efficiency would not be very informative in identifying species. in generating patterns of banding that are unique to the species and second in exhibiting or lacking unique band (s) that distinguish an individual from the rest of the population (Dhahi et al., 2011)   This technique uses random primers under low specificity conditions; it is less costly and faster and easier to perform than analogous systems, either phenotypic or genotypic. (Loutit and Tompkins, 1991) Polymorphisms at DNA level may occur as a result of several types of mutations, such as single base change in the primer-annealing site in the genome that prevents amplification by introducing a mismatch at 3 end of a DNA segment (Hurtado and Rodriguwz, 1999). Other sources of polymorphisms may include deletion of a priming site, insertion that render priming sites to be too distant to support amplification, or they may change the size of DNA segment without preventing its amplification (Williams et al., 1990).
Multidrug resistance (MDR) is a common feature of P. aeruginosa strains. Rapid identification of the organism is necessary to expedite the onset of treatment and to reduce the duration and dosage of antibiotic therapy, which in turn could minimize the development of antibiotic resistance. (Jamasbi and Poudfoot, 2008) A significant decline in susceptibility of P. aeruginosa to B-lactams, aminoglycosides, and quinolones has been reported, Nosocomial outbreaks MDR in different hospitals have appeared that the frequency and rate of resistance to individual antibiotics are different in different genotype (Gales et al., 2001).
The differences in antibiotic susceptibility of P. aeruginosa in different genetic variations could be attributed to the differences in patient population, the duration of hospitalization, cross-infection, and the dose and types of antibiotics. Transmission of resistant fragments within smaller hospital units may be responsible for this phenomenon, and the determination of antimicrobial profiles is another typing method used frequently as a supplemental epidemiological tool for strain differentiation of P. aeruginosa. It should be noted, however, that antibiotic susceptibility profiles are less stable than O-antigenic markers, given the resistance factors that can occur under pressure of antibiotic therapy. (Jamasbi and Poudfoot, 2008).