Molecular characterization of Pseudomonas aeruginosa isolates from Sudanese patients: A cross-sectional study

Introduction

Pseudomonas aeruginosa is a gram-negative bacterium that is found widely in the environment and engages in various forms of interactions with eukaryotic host organisms. It is an opportunistic pathogen that is widely spread in humans, giving rise to a broad spectrum of infections in community and healthcare facilities^1,2. Due to the extended spread of P. aeruginosa habitat, the control of the organism in a hospital setting is very difficult, and makes it practically impossible to prevent contamination³. The major threat is the infection of patients who are immunocompromised or those in burns, neonatal and cancer wards^4,5. Infection of P. aeruginosa is still one of the main causes of death among the critically ill and patients with impaired immune systems in spite of the development of newer and stronger antibiotics.

Sequencing of 16S rRNA worldwide has provided interesting and useful information^6–9. For instance, with the use of 16S rDNA sequencing, 215 novel bacterial species, 29 of which belong to novel genera, have been discovered from human specimens in the past 7 years of the 21st century (2001–2007). One hundred of the 215 novel species, 15 belonging to novel genera, have been found in four or more subjects¹⁰. In Sudan, there is deficient data on sequencing about bacteria; therefore it is important to investigate what kinds of bacteria affect Sudanese people. Consequently, the objective of this study was to isolate and characterize P. aeruginosa from samples obtained from Sudanese patients by sequencing the 16SrRNA gene.

Methods

Conventional polymerase chain reaction (PCR)

All bacterial genomic DNA were used as templates for PCR amplification of the 16S rRNA gene. The two primers used were 27F (5ʹ AGAGTTTGATCCTGGCTCAG-3ʹ) and 1495R (5ʹ CTACGGCTACCTTGTTACGA- 3ʹ) for forward primer and reverse primer, respectively (Macrogen, South Korea). The 25μL PCR reaction mixture (Intro Biotechnology, South Korea) contained 1μL DNA, 1x reaction buffer (10x) with 3mM MgCl₂, 2.5U i-Taq^TM DNA polymerase (5 U/μL), 2.5mM dNTPs, 1μL of 10 pmol of each primer, and 1x of gel loading buffer, followed by completing the volume to 25μL with nuclease free water. Thermal cycling conditions were as follows: 94°C for 5 min; 30 cycles of denaturation (94°C for 1 min), annealing (58°C for 1 min), extension (72°C for 2 min); final extension at 72°C for 10 min. PCR was performed on a Bio-Rad (DNA engine/Dyad Peltier) automatic thermal cycler. Duplicate PCR of every sample were carried out for confirmation^11,12. PCR products (5μl) were analyzed by gel electrophoresis in 1.0% agarose stained with ethidium bromide. The results were photographed under ultraviolet light machine (Transillumnator; Uvite, UK) to detect the specific amplified product by comparing it with 100 base pairs standard DNA ladder (Figure 1) and the remains from PCR products were store at -20°C until sequencing.

Figure 1. Agarose gel electrophoresis of PCR products after amplification of the 16S rRNA gene.

Lanes: 1- 8 positives 16SrRNA gene molecular weight marker (O’Range Ruler 100 DNA Ladder, SM1143-Fermentas).

Results

A total of 40 clinical isolates were identified as P. aeruginosa by conventional methods, including growth characteristics, colony morphology, and biochemical tests. The results revealed that P. aeruginosa were dominant in wound infection cultures (42.5%), followed by (32.5%) from urine cultures and (25%) from sputum cultures.

The 16SrRNA in the isolates was amplified by PCR. In total, 20 were sent for characterization by sequencing of PCR products, 5 out of these 20 had a clear chromatogram, leading to further sequence analysis. Sequence analysis by BLASTn revealed 100% identity with P. aeruginosa from Iran (KX108935.1), China (KT943978.1), Australia (JN609194.1), Austria (KX214108.1), India (KU672378.1) and South Africa (KU764451.1), and slightly different from a sample from Australia (FJ648815): there was one substitution of A to G in position 148 (Figure 2). The phylogenetic tree of the 16S rRNA gene and other 16S rRNA genes obtained from the database revealed that the tree is classified into two branches. All isolates have a common ancestor. The isolate 120 is at the upper branch, isolate 113 and isolate 114 are sister groups, as shown in Figure 3.

Figure 2. BioEdit multiple sequence alignment of the five isolates and other selected strains.

The isolates had 100% identify with samples from Iran, China, Austria, India, and South Africa. There was one substitution of A to G in position 148 from a sample from Australia (FJ648815).

Figure 3. Phylogenetic tree of the 16S rRNA gene and other 16S rRNA genes.

The tree is classified into two branches. All isolates have a common ancestor. The isolate 120 is at the upper branch, isolate 113 and isolate 114 are sister groups.

Discussion

P. aeruginosa can cause various clinical infections. In the present study, 42.5% of isolates were identified from wound infection. Beside the highly preserved conserved primer binding sites, 16S rRNA gene sequences include hypervariable regions with high conservation that have the ability to characterize species-specific signature sequences beneficial to the characterization of bacteria^17,18. The present results agree with Didelot et al., who reported that 16S rRNA gene sequencing is now common in medical microbiology as a quick and inexpensive alternative to phenotypic approaches of bacterial identification¹⁹. We investigated the phylogenetic affiliation of the pseudomonads and reexamined the 16S rRNA sequence data available in public databases. According to an alignment of 16S rRNA sequences, we identified P. aeruginosa-specific signature sequences.

16S rRNA sequencing can be used to identify genetically atypical P. aeruginosa isolates from different origins. Our observation of 40 isolates from 40 samples, further showed the utility of 16s rRNA PCR amplification. This reveals the high specify of the primers and accuracy of the PCR. Sequencing of the 16S (rRNA) gene was used as an effective tool for determining phylogenetic relationships between bacteria. The features of this molecular target make it valuable and useful for bacterial detection and identification in the clinical laboratory.

Conclusions

In conclusion, 16S rRNA-based PCR assay and sequencing is highly specific, sensitive and reliable for identification of P. aeruginosa and its differentiation from other genotypic closely related Pseudomonas species. DNA sequencing of the 16S rRNA gene has been used as an effective tool to study bacterial phylogeny and taxonomy relationships between bacteria and for bacterial detection.

Data availability

The results of the nucleotide sequences of the 16S rRNA gene were submitted in the GenBank database. Accession numbers: KX650648, KX650649, KX650650, KX650651 and KX650652.