Molecular characterization of multidrug resistant (MDR) clinical isolates of Pseudomonas aeruginosa from Nsukka, South Eastern Nigeria

Background P. aeruginosa is an important nosocomial pathogen with increasing resistance to antibiotics. Objective: This study was performed to evaluate the genetic relatedness of MDR clinical isolates of P. aeruginosa. Method A total of 1000 samples were analysed in the study. Antibiotic resistance profiles of the isolates were determined using Kirby Bauer disk diffusion method. Polymerase chain reaction (PCR) and sequencing were simultaneously used to detect the consensus region of 16S rRNA. Genetic relatedness of the isolates was determined using restriction patterns from ALU 1 digest and random amplified polymorphic DNA. Results Out of the 192 P. aeruginosa isolates recovered, 136 (78.83%) were multidrug resistant. Sequence analysis of the confirmed isolates (80.68%) revealed that all the isolates shared homology with each other and also showed sequence similarity to known strains of P. aeruginosa (ATCC 27853; KT 315654; KU 321274 and KT894767). The PCR-Restriction fragment length polymorphism (PCR-RFLP) analysis revealed that there was a lot of genetic relatedness among the isolates. The RFLP finger printing technique detected seven distinct RFLP types among the isolates. Conclusions Thus, study shows that there is high prevalence of MDRPA and high degree of genetic relatedness among the MDRPA isolates circulating in Nsukka area.


Introduction
P. aeruginosa has emerged as one of the most problematic nosocomial pathogens.It is considered as a major opportunistic pathogen that causes infection in immune depressed individuals, burn patients or cystic fibrosis patients 1 .
It is responsible for various nosocomial infections, including those of the blood, lungs, wound, ear and urinary tract 2,3 .These infections are often difficult to eradicate due to the resistance of P. aeruginosa to many antibiotics 4 .It is ubiquitous in nature and its ability to survive in moist environment with innate resistance to many anti-biotics and antiseptics enable it to constitute a common pathogen in hospitals, particularly in Intensive Care Units (ICUs).The increasing use of antibiotics and the growing numbers of invasive procedures, together with the development of intrinsic and acquired resistance mechanism of P. aeruginosa, cause the evolution of numerous multidrug resistant P. aeruginosa (MDRPA) outbreak in clinical settings 5 .Multidrug resistant P. aeruginosa is a public health problem that affects many countries of the world.
A rapid and accurate system for the identification of P. aeruginosa is important to isolate patients and prevent further spreading of the diseases.Conventional biological typing methods such as biotyping, phage typing, serotyping and bacteriocin (pyocin) are well established and have been applied to the identification of P. aeruginosa infections but may lack discriminatory power and stability.
Many studies are satisfied by using the API 20 test system or classical biochemical test for bacterial identification 6 .
However, P. aeruginosa adaptive ability causes difficulties for the sensitivity of these methods.Therefore, it has become necessary to develop genotype-based characterization systems capable of accurately identifying these bacteria despite any phenotypic modifications.Molecular identification eliminates the problem of variable phenotype and allows for more accurate identification of bacteria 7 .16S rRNA genes are highly conserved among all organisms and they possess various unique species-specific regions that allow for bacterial identification.Polymerase Chain Reaction (PCR) is highly sensitive, specific and rapid method which vastly improved the detection of P. aeruginosa especially when using species-specific primer for 16S rRNA 8 .Sequencing of 16S rRNA is a molecular technique for characterization of bacteria and tools used to analyse the phylogenetic relationship of an organism because of their information content, conservative nature, and universal distribution 9 .The use of DNA-based typing methods is becoming increasingly important in epidemiological survey and differentiation of Pseudomonas species.Several molecular typing techniques such as multilocus sequence typing, DNA microarray and indirect method of sequence analysis such as Restriction Fragment Length Polymorphism (RFLP); Random Amplified Polymorphic DNA (RAPD) and Pulsed Field Gel Electrophoresis (PFGE) have been described to differentiate between the isolates and clonal groups of P. aeruginosa 10,11 .Restriction fragment length polymorphism refers to the polymorphic nature of the locations of restriction enzyme site within defined genetic regions.Specific genetic loci are routinely amplified and examined for differences indicative of strain variation.The specific locus to be examined is amplified with gene-specific primers and is subjected to RFLP analysis.It is a reliable and relatively simple method but prior knowledge of the DNA sequence is necessary.On the other hand, Random Amplified Polymorphic DNA (RAPD) does not require any specific knowledge of the target DNA sequence, making it a flexible and powerful tool with general applicability.These typing techniques (RFLP & RAPD) are useful for establishing clonal relationship between individual isolates in hospital settings and are therefore important to recognize nosocomial transmission and guide infection control practice.These molecular typing techniques have been widely used in developed countries.However, there is scanty information on the genetic study of P. aeruginosa in Nigeria, particularly in Enugu state.This study there-fore aimed to isolate MDR clinical isolates of P. aeruginosa; characterize using PCR and sequencing and to elucidate their genetic relatedness using PCR-RFLP and RAPD. ) and an informed consent was obtained from patients.Wound/pus swabs, ear swabs, sputum and swabs from hospital fomites were inoculated on brain heart infusion broth (Oxoid, UK) incubated for 24 h at 37 0 C. The next day, a loopful of the broth culture of each sample was inoculated onto sterile Pseudomonas cetrimide agar (Oxoid, U.K) which was supplemented with 10 ml/L of glycerol and incubated at 370C for 24 h.Urine samples were inoculated directly onto Pseudomonas cetrimide agar plate.Colonies were examined for the presence of blue-green or yellow-green pigments.Thereafter, the isolates were characterized by standard microbiological methods using P. aeruginosa ATCC 27853 as a control strain.

Genomic DNA Extraction
The genomic DNA extraction was performed using zymo research fungal/bacterial DNA mini prep kit (Zymo Research, USA) according to the manufacturer's protocol.The purity of the extracted DNA was determined by checking the absorbance at 260 and 280 nm using Nanodrop Spectrophotometer.

DNA Amplification
The extracted DNA was amplified with 16S rRNA primer targeting P. aeruginosa consensus region (Inqaba Biotechnical Company, South Africa) (Table 1

Analysis of PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) and Random Amplified Polymorphic DNA (RAPD-PCR)
Numerical Taxonomy System for Personal Computer (NTSYS-pc) v.2.20 software was used to analyse the band profiles.Dendrogram for cluster analysis of the strains were constructed using the Un-weighted pair group method using arithmetic average (UPGMA)

Results
Out of 1000 samples collected, P. areugionsa was recovered in 19.20% (192), comprising 136 (78.85%) multidrug resistant strains.The percentage occurrence of P. areugniosa isolates from different clinical samples was highest in wound/pus (32.86%); followed by ear swabs (22.50%); sputum (12.00%); hospital fomites (8.00%) and urine (5.00%).Analysis of the DNA purity using the ratios of absorbance at 260 and 280nm (A 260 /A 280 ) showed relatively pure DNA samples with A 260/280 values between 1.85 and 1.93.Quantitative analyses showed that the samples contained about 134-645 ng/µl DNA.The result of the amplification using the primer pair targeting the consensus region of bacterial 16S rRNA gene are presented in Figures 1.Approximately 80.68 % of the P. aeruginosa isolates expressed positive amplification of the 1499 bp of 16S rRNA gene, which confirmed the assumption that they were strains of P. aeruginosa.The 16S rRNA gene region of these isolates were sequenced, aligned, and blasted and sequences analysis revealed that all the isolates shared homology with each other and also showed sequence similarity to known strains of P. aeruginosa (Figure 2).    2. The present study agrees with the study carried out by Amutha and Kokila, who detected that after blast analysis all the P. aeruginosa isolates gave 99% similarity and the phylogenetic tree generated by NCBI tool proves that this organism is genetically related 20 .Since determining bacterial genetic relatedness is essential for cross-infection evaluation, different genotyping methods have been established 21,22 .In this study, PCR-Restriction fragment length polymorphism (PCR-RLFP) and Random amplified polymorphic DNA (RAPD) techniques were used to determine the genetic relatedness among multidrug resistant P. areuginosa isolates.PCR-Restriction fragment length polymorphism technique could be considered as a simple and sensitive method for detection and identification of bacteria.It has a high degree of discriminatory capability.In this study, the endonuclease digestion of 16S rRNA gene products generated different fragments ranging from 100 -800 bp (Figure 3).
The analysis of the PCR-RFLP revealed that there was a lot of genetic relatedness among the isolates.The RFLP typing technique detected seven (7) clones/distinct RFLP types among the multidrug resistant isolates.Random amplified polymorphic DNA (RAPD -PCR) technique has been shown to be useful technique to study the genetic variability between bacterial strains including P. aeruginosa due to its great specificity and sensitivity 23 .Moreover, RAPD-PCR could be considered as a source for tracking the infections since it is cost effective PCR based technique.In this study, DNA fingerprinting was performed using three oligonucleotide (RAPD) primers (M13, B-01 and B-11).It was observed that only the M13 primer was able to detect polymorphism among the isolates.The phylogenetic tree generated shows that these organisms are genetically related (figure 5).
The RAPD technique detected three (3) RAPD patterns.This finding is in line with the work of Husch who used the same M13 primer for RAPD-PCR fingerprinting for MDR P. areuginosa isolates obtained from intensive care Burn unit, and found that MDRPA isolates possessed identical RAPD patterns 14 .Our results demonstrate that most of the isolates probably originated from the patients themselves; however, cross-infection of P. areuginosa between patients is possible to occur, suggesting nosocomial infection control problem.

Conclusion
The present study, highlights that there is an alarming increase of clinical infections caused by multidrug resistant strain of P. aeruginosa.The PCR-RFLP and RADP analysis revealed that there was a lot of genetic relatedness among the isolates.The extensive use of broad spectrum of antimicrobial agents in various hospitals were probably responsible for the emergence and selection of this multidrug-resistant strains.This calls for rational drug use and need for improved personal and environmental hygiene especially within the hospital settings.

Figure 2 :
Figure 2: Phylogenetic tree showing 100% similarity among the P. aeruginosa isolates and comparison by the other Pseudomonas strains based on their 16SrRNA sequence

Figure 4 :
Figure 4: Dendrogram generated from the PCR-RFLP showing genetic relatedness among MDRPA isolates.W=wound; E=Ear swabs; and U= Urine A total of 1000 samples comprising 950 clinical samples and 50 samples from hospital fomites were collected from various hospitals in Nsukka, South Eastern Nigeria between March and October, 2019.Of the 950 clinical samples, 36.84%(350) were wound/pus; 31.58%(300) were urine; 21.05% (200) were Ear swabs and 10.53% (100) were sputum.The study was approved by the Ethics Committee of the Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka.(ECC reference no: FRSRE/UNN/19/0008

Table 1 :
06% GEPAL CA-630, 0.05% Tween 20, 25 units / ml Taq DNA polymerase (BioLab, England), 0.5 µl (10 µM) each of the forward and reverse primers (Inqaba biotech, South Africa), 5 µl of the extracted DNA and 6.5 µl of sterile Nuclease-free water ( Norgen biotech corp.Canada).This was vortexed at low speed and placed in a thermal cycler (BIBBY) -Scientific Ltd, UK.The PCR amplification program for the primers used is shown in Table1.The PCR products were resolved on 1.5% agarose gel, stained with ethidium bromide (0.5 µg/ ml) and electrophoresis was carried out at 70 volts for 90 min and visualized under UV transilluminator.A 100 bp DNA ladder (Norgen Biotek Corp, Canada) was used as DNA molecular weight marker.Primer Sequences and PCR Conditions Used dard buffer.The PCR reaction mixture was prepared in a 25 µl reaction volume containing 12.5 µl of 1X Master mix with standard buffer, 20 mM Tris-Hcl, 1.8 mM MgCl 2 , 22 mM NH 4 Cl, 22 mM KCl, 0.2 mM DNTPS, 5% glycerol, 0.DNA SequencingThe PCR amplicons of 16S rRNA primer were subjected to DNA sequencing by Inqaba Biotech Sequencing Service, South Africa.The sequencing was done with the ABIV 3.1 Big dye kit according to the manufacturer's instructions (http://mvz.berkeley.edu/egl/inserts/Big-DyeV3.1Protocolmannualpdf.) and the labelled products were then cleaned with zymoseq clean-up kit http://www.African Health Sciences, Vol 23 Issue 3, September, 2023