Molecular characterization of Pseudomonas aeruginosa isolates from Sudanese patients: A cross-sectional study [version peer review: 1 approved with reservations, 1 not approved]

Background: 16S rRNA gene sequence analysis is a robust tool for characterization of new pathogens in clinical specimens with suspected bacterial disease. The aim of this study was to characterize Pseudomonas aeruginosa isolated from clinical specimens by sequencing the 16S rRNA gene. Methods: Forty bacterial isolates were obtained from different clinical specimens (wound, urine and sputum) using enrichment selective media and biochemical tests to characterize and identify the bacteria as P. aeruginosa. DNA was extracted from P. aeruginosa using the Chelex method. A universal primer was used to amplify 16S rRNA genes by a conventional PCR technique. The amplified PCR products were sequenced, and the sequences were viewed by Finch TV program version 1.4.0. The identity and similarity of the nucleotide sequence of the isolated strains was detected by comparing them with published sequences using BLASTn. Phylogenetic trees were constructed using Phylogeny.fr software. Results: Sequence analysis by BLASTn Manuscript deals with the characterizarion of Pseudomonas clinical isolates by PCR and 16S rRNA gene sequencing. Only twenty isolates were used for sequence comparison, and actualy only five sequences are compared by BLAST and deposited on Genbank DATABASE. Authors claim that sequencing of 16S rRNA gene is useful for Pseudomonas identification, but this fact has been shown in hundred of papers. It is not a novelty. Moreover, no other molecular analysis is done with the isolates, as for example, identification of resistance mechanisms. In addition, isolates were already identified as Psedomonas aeruginosa by biochemical methods. A more useful analysis would be to take the full number of isolates and without previous identification, characterize them as P. aeruginosa.


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 contamination 3 . 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)(2002)(2003)(2004)(2005)(2006)(2007). One hundred of the 215 novel species, 15 belonging to novel genera, have been found in four or more subjects 10 . 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.

Clinical isolates
This was cross sectional laboratory based study, conducted in Khartoum state in the period from January to April 2016. The study was approved by the Ethical and Scientific Committee of the Medicinal and Aromatic Plants and Traditional Medicine Research Institute, National Center of Research, Khartoum, Sudan (approval number 03-16) which ensures that all ethical considerations for conducting the research in a way that protects patient's confidentiality and privacy are followed. Informed consent was obtained from the hospital laboratories (laboratory manager) after providing them with the ethical approval to collect samples during routine procedures from the microbiology laboratories. Participants' privacy and confidentially was protected for all samples; personal information was not of great value in the current study and was thus not taken. Consequently, the Ethical and Scientific Committee waived the need for patient consent.
A total of 40 isolates of P. aeruginosa (all samples available) were obtained from three hospitals in Khartoum State (Al Ribat Hospital, Bahri Hospital and Souba Hospital).
The bacterial isolates were collected from sputum culture, urine culture and wound infection because these types of cultures were dominant. Standard biochemical tests 7 were performed on all samples for the isolation and identification of the bacterial isolates and were performed at the Medicinal and Aromatic Plants and Traditional Medicine Research Institute (MAPTMRI), Department of Microbiology. P. aeruginosa presence was confirmed in all 40 samples. All media required for biochemical tests were obtained from LAB M, UK and Laboratories Flow Media, Sudan.

DNA extraction
Bacterial DNA was isolated by the Chelex-based protocol 7 for all samples without deviation from the methodology.
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 (5´ AGAGTTTGATCCTGGCTCAG-3´) and 1495R (5´ CTACGGCTACCTTGTTACGA-3´) 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 MgCl 2 , 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.
Sequencing of 16S rRNA gene Isolates were packaged according to the International Air Transport Association guidelines and shipped with authorized permission to Macrogen Company (Seoul, South Korea). Purification and standard forward sequencing of 16S rRNA were done by ABI Genetic Analyzer (Applied Biosystems). We randomly selected 20 isolates only for DNA sequencing due to the limitation of resources.

Bioinformatics analysis
The chromatogram sequences were visualized using Finch TV program version 1.4.0 13 . The nucleotide sequences of the 16S rRNA gene were searched for sequences similarity using online BLASTn 14 . Highly similar sequences (accession numbers KX108935.1, KT943978.1, JN609194.1, KX214108.1, KU672378.1, KU764451.1 and FJ648815.1) were retrieved from NCBI GenBank and subjected to multiple sequence alignment using BioEdit software version 7.2.5. Newick format was withdrawn from ClustalW 12 , to create phylogenetic trees in Phylogeny.fr software 15 . MEGA6 software version 0.06 was used for confirmation of phylogentic trees 16 .

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. KX108935

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 identification 19 . 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.

Competing interests
No competing interests were disclosed.

Grant information
The author(s) declared that no grants were involved in supporting this work. Partly