Sequence Analysis of Novel Genes in Clinical and Environmental Pseudomonas aeruginosa Iraqi Isolates.
MATERIALS AND METHODS
Out of 100 samples, 30 P. aeruginosa isolates were recovered from clinical and environmental sources. These samples include 21 isolates from burn wound, 9 isolates from the hospital environment (antiseptics and soaps in burn unit). All samples and patients were obtained at Al-Hilla Surgical Teaching Hospital in Al-Hilla City/Iraq.
Diagnosis of bacteria
All samples that collected from different sources were cultured on MacConkey agar and incubated overnight at 37[degrees]C of plates. Diagnosis of bacteria was carried out by biochemical methods (oxidase, IMVIC, and nitrate reduction), as well as using phenazine production according to previous studies (7, 8).
DNA of 26 bacterial isolates collected from 21 foot ulcer and 5 environmental samples was extracted according to the Geneaid manufacturer's instructions (Presto TM Mini Gdna bacterial Kit).
Molecular detection of phenazine modifying genes by PCR
Primers, PCR program, and mixtures were used to detectphzM and phzS genes as following; each 25 [micro]l of PCR mixture consists of forward and reverse primer (2.5 [micro]l). DNA extraction in a concentration of 0.1[micro]g/ml (5 [micro]l) and mastermix (12.5 [micro]l). The polymerase chain reaction product was detected by gel electrophoresis on 1.5% agarose gel for 40 min. at 70V. Upstream and downstream primers for phzM and phzS genes were used according to [9, 10] respectively.
Sequencing of phzm and phzs genes and sequence alignment
Four isolates of P. aeruginosa that carry both genes phz M and phzS were subjected to sequencing in this study. According to the results of PCR products, the products were separated on a 2% gel electrophoresis agarose and visualized by UV light at (302 nm) after addition of Red Stain or ethidium bromide. Automatic sequencing was conducted in a biotechnology lab using DNA sequencer 3730XL, Applied Biosystems. The Basic Local Alignment Search Tool (BLAST) and BioEdit programs using the National Center Biotechnology Information (NCBI) at (http:// www.ncbi.nlm.nih.gov) were used to analyze this data.
E value and score
The expectation value is defined to give an estimate of the number of times expected to get the same similarity coincidental and the lower the value of E. This indicates that the degree of similarity was high between sequences which give greater confidence. The value of close to zero means that these sequences are identical and the bit score: a statistical analysis of the moral similarity and the higher value indicate that there is a high degree of similarity. If the value dropped from the class of 50 points, this similarity may not be shown.
RESULTS AND DISCUSSION
Detection of phenazine modifying genes by PCR
The two novel genes, phzM and phzS which are responsible for pyocyanin pigment synthesis in P.aeruginosa. In the present study, the PCR technique was used to detect phzM and phzS genes through the use of pieces of DNA with a limited number of the oligonucleotide which acts as primer specialized virulence genes of P. aeruginosa. It was found that most of the clinical isolates were carried the genes responsible for pigment production with some exclusion. The results also found that two environmental isolates lacked both phenazine genes examined. Phenotypically, all the clinical isolates were found to be producing phenazine, but the environmental isolates were not be the producer. Regarding phzM, out of 26 P. aeuroginosa isolates, 21 (80.76%) isolates harbored this gene at 330 bp in PCR amplification while phzS gene represents 14(53.84%) with 664 bp, as shown in (Fig.1). The findings of the present study were comparable with that obtained by (9), which initiates that phzM gene exists at a percentage of (84%) in P. aeruginosa isolates. However, similar findings were obtained by (4), which referred to the presence of the two genes is essential to create phenazine in P. aeruginosa. Although, the phzM gene regulating phenazine synthesis was found in (80.76%) of present tested isolates, that may be due to the diversity of these isolates (11). According to mutant strain analysis of these two genes, it was postulated that mutant strain demonstrates variable phenazine production (4).
Sequencing of phzm and phzs genes
In the present study genotypic variation of environmental and clinical isolates of this bacterium was studied by sequencing of phzM and phzS genes. The relationships of genetic evolution of pseudomonas isolates were examined by comparing the analysis of the sequence with the NCBI. On the basis of the evidence by sequencing, it has been postulated that all the four isolates from clinical and environmental samples belong to the species Pseudomonas aeruginosa (Fig.2). The analysis of phylogenetic sequences has not revealed considerable diversity in environmental and clinical isolates (Fig.2). The findings of phzM gene sequence demonstrated that there were 7 mutations with 99% identities in 4 isolates as shown in (table 1) and (Fig.3), While for the phzS gene 2 mutations with identities 99% were detected by sequencing in the same isolates (table 2) and (Fig.4). The current results also appeared that P. aeruginosa isolates collected from environmental and clinical sources have a core genome which was highly conserved. However, these isolates tended to be variable in regards to the presence of regions involved in the phenazine phenotype.
Effect of mutations in phzM and phzS genes on phenazine production
To assess the impact of the biosynthesis modifying genes phzM and phzS on phenazine production in pseudomonas isolates with mutations in these genes. The isolates showed uncommon pigment phenotypes when they were cultured. While blue cultures of wild-type were attributable to the pyocyanin production, this finding was in agreement with the results conducted by (4). It was interesting to mention that the existence of dual operons regulating biosynthesis of phenazine makes this bacterium more flexibility in modulating the number of phenazine compounds. It has been suggested that the variation in phenazine production might be ascribed to growth phase or in response to signals from the environment. Mutations affect the phzM and phzS genes through the creation of change in the gene sequence. The results in the table (1) showed that 1(11.11%) nonsense mutation which leads to change the code of amino acid stop codon, causing dysfunction of the protein. Furthermore, there is 8(88.88%) missense mutation in both genes, this type of mutation influences the phenotype because they lead to substitution of amino acids and thus in protein. An amino acid can be replaced with another amino acid that has similar chemical characteristics, therefore the protein could normally work. There is also an amino acid encoded by more than one code which could result in mutation. Nevertheless, this mutation does not produce any change in the translation.
From the present study, it seems that P. aeruginosa isolates of clinical and environmental sources have a very preserved genome core. The sequence of bacterial genome gives significant evidence to appreciate the regulatory and metabolic network that link chromosomal genes. Although the pseudomonas isolates have mutations in phzM and phzS genes, it has been proposed that these mutations have no role in the pathogenesis. In this site, the crucial part of P. aeruginosa virulence looks to be the regulation of gene expression instead of the existence or nonexistence of genes.
I am thankful the Microbiology Department, Medicine College of Babylon University/Iraq for the facilities provided in the completion of the work. I am also thankful Hiba Jasim for her cooperation.
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Huda Hadi Al-Hasnawy
The University of Babylon, College of Medicine, Department of Microbiology, Iraq.
(Received: 08 January 2018; accepted: 20 February 2018)
* To whom all correspondence should be addressed.
Caption: Fig. 1. A: Electrophoresis gel of PCR product of phzM genes. Lane L: DNA marker (100-1500bp); Lane (24) negative control; Lane (22) environmental isolate; Lane (5, 12, 21, 25, 26) clinical isolates give a positive result for this gene. B: Gel electrophoresis of PCR product of the phzS gene. Lane L: DNA marker (100-1500bp); Lane (24) negative control; Lane (3, 22) environmental isolates; Lane (2, 14, 15, 23) clinical isolates
Caption: Fig. 2. Phylogenetic tree based on 16S rRNA gene sequence of 4 isolates of Pseudomonas aeruginosa compared with sequence available in the GenBank. Nodes indicate E value
Caption: Fig. 3. Sequencing results of phzM genes of Pseudomonas aeruginosa in 4 isolates
Caption: Fig. 4. Sequencing results of phzS genes of Pseudomonas aeruginosa in 4 isolates
Table 1. Type of mutations in the phzM gene sequence of Pseudomonas aeruginosa in 4 isolates No. Of Wild- Mutant Type of Location sample type type mutation 5 GGC GGA Transversion 784321 GAT TAG Transvertion 784322 GAT TAG Transvertion 784324 ATC TTC Transvertion 784342 12 21 25 AAC TAC Transvertion 784316 GAT ATT Transition 784322 GAT ATT Transvertion 784323 No. Of Wild- Amino acid change sample type 5 GGC Glycine > Glycine GAT Aspartic acid > Stop codons GAT Aspartic acid > Stop codons ATC Isoleucine> Phenylalanine 12 21 25 AAC Asparagine> Tyrosine GAT Aspartic acid > Isoleucine GAT Aspartic acid > Isoleucine No. Of Wild- effect Sequence ID Identities sample type 5 GGC nonsense ID: CP020603.1 99% GAT Missense GAT Missense ATC Missense 12 ID: CP020603.1 100% 21 ID: CP020603.1 100% 25 AAC Missense ID: CP020603.1 99% GAT Missense GAT Missense Table 2. Type of mutations in the phzS gene sequence of Pseudomonas aeruginosa in 4 isolates No. Of Wild- Mutant Type of Location Amino acid change sample type type mutation 2 GCC ACC Transition 8660 Alanine> Threonine CGC CAC Transition 8697 Arginine> Histidine 14 15 23 No. Of Wild- effect Sequence ID Identities sample type 2 GCC Missense ID: KX180139.1 99% CGC Missense 14 ID: CP004061.1 100% 15 ID: CP008739.2 100% 23 ID: CP004061.1 100% Table 3. Effect of the type and percentage of mutation on phzM, phzS genes Effect of mutation number phzM (%) phzS (%) percentage nonsense 1 1(11.11) 0 (0.00) 11.11 Missense 8 6 (66.66) 2 (22.22) 88.88 Total 9 7 2 99.99
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|Author:||Al-Hasnawy, Huda Hadi|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Mar 1, 2018|
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