Phenotypic And Molecular Characterizations of Pseudomonas Aeruginosa Isolated Among Patients Admitted at Tikur Anbessa Specialized Hospital and Yekatit 12 Hospital Medical College, Addis Ababa, Ethiopia
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Date
2025-11-07
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Addis Ababa University
Abstract
Background: Pseudomonas Aeruginosa Is a Leading Cause of Hospital-Acquired Infections and Is Increasingly Resistant to Multiple Antibiotics, Which Complicates Treatment and Infection Control. Its Ability to Form Biofilms, Express Multiple Virulence Factors, And Harbor Diverse Molecular Mechanisms of Antimicrobial Resistance Contributes to Its Persistence and Pathogenicity in Healthcare Settings. Investigating Biofilm Formation, Virulence Traits, And Molecular Resistance Mechanisms of P. Aeruginosa Is Essential for Understanding Its Pathogenic Potential, Informing Effective Infection Prevention Strategies, And Guiding Clinical Management.
Objectives: This Study Aimed to Determine the Phenotypic and Molecular Characterizations of P Aeruginosa Among Patients Admitted at Tikur Anbessa Specialized Hospital and Yekatit 12 Hospital Medical College, Addis Ababa, Ethiopia.
Methods: A Cross-Sectional Study Was Conducted from August 2022 to September 2025. A Total Of 422 Clinical Specimens Including Blood, Urine, Burn Swabs, And Surgical Site Wound Swabs Were Collected from Hospitalized Patients at Tikur Anbessa Specialized Hospital (TASH) And Yekatit 12 Hospital Medical College (Y12HMC). Eighty-Four Isolates Identified as Pseudomonas Species Were Stored in STGG Medium At −80°C And Later Shipped to Uppsala University, Sweden, For Molecular Characterization.
Isolation And Identification of P. Aeruginosa Were Performed Using Standard Microbiological Techniques. Antimicrobial Susceptibility Testing Was Conducted Against 11 Antibiotics Using the Kirby-Bauer Disk Diffusion Method According to CLSI Guidelines. Carbapenem Susceptibility Was Determined by Broth Microdilution, And Colistin Susceptibility Was Tested Using the Colistin Broth Disk Elution Method. Biofilm Formation Was Assessed Using the Microtiter Plate Assay. Whole-Genome Sequencing (Illumina Hiseq 2500) Was Performed on the 84 Isolates, And Downstream Genomic Analysis Was Conducted On 64 Isolates to Characterize Virulence Factors, Serotypes, Antibiotic Resistance Determinants, And Phylogenetic Relationships. Bivariate And Multivariable Logistic Regression Analyses Were Used to Identify Factors Associated with Multidrug Resistance (MDR). The Spearman Correlation Coefficient Was Applied to Evaluate the Relationship Between Biofilm Formation and Antimicrobial Resistance.
Results: The Overall Prevalence Rate of Pseudomonas Aeruginosa Among The 422 Clinical Specimens Was 19.6% (83/422). The Distribution of Isolates Across Hospitals Was 43.4% (N=36) From Y12HMC And 56.6% (N=47) From TASH. Among The Positive Isolates, The Distribution by Specimen Type Was as Follows: Urine 32.5% (27/84), Blood 22.9% (19/84), Surgical Site Wounds 27.7% (23/84), And Burn Wounds 16.9% (14/84). High Resistance Rates Were Observed for Ciprofloxacin (51.8%), Ceftazidime (50.6%), And Cefepime (48.2%), While Low Resistance Was Noted for Ceftazidime-Avibactam (4.8%) And Imipenem (16.9%). The Overall Proportion of Multidrug-Resistant (MDR) Isolates Was 56.6%. Biofilm Production Was Detected In 95.2% Of Isolates, with 27.7% Classified as Strong And 39.8% As Moderate Biofilm Producers. A Significant Positive Correlation Was Observed Between Biofilm Formation and Multidrug Resistance. Previous Exposure to Ciprofloxacin Was Identified as an Independent Factor Associated with MDR P. Aeruginosa. Among The 64 Isolates Subjected to Whole-Genome Sequencing, Eight Serotypes Were Identified. The Most Prevalent Serotypes Were O6 (50%), O11 (14.1%), O3 (10.9%), O5 (9.4%), O1 (7.8%), O2 (3.1%), O4 (3.1%), And O9 (1.6%). Serotype O6 Was Most Frequent Across All Infection Types, While O1, O3, And O11 Predominated in Specific Specimens. Serotype-Specific MDR Rates Varied: O11 Had the Highest MDR Rate (88.9%), Followed by O5 (66.7%); O6 And O2 Had Equal Distributions of MDR And Non-MDR Isolates (50% Each), Whereas O4 And O9 Were Entirely Non-MDR (100%). Amongst The 241 Virulence Genes Identified, 83.4% Were Present in Nearly Every Isolate. The Virulence Genes Identified Were Flagella-Related Genes, Biofilm Related Genes, Secretion System Components, Secreted Factors, And Toxin Related Genes. The Most Common Toxins Identified Were Exoy (96.9%), Exot (96.8%), Exos (95.3%), Toxa (93.8%) And Exou+ (6.3%). There Were Four (6.3%) Exou+ Strains and One (1.6%) Exou+Exos+ Multidrug Resistant Strain, All of Which Were O11 Serotypes. The Most Frequently Detected ESBL Gene Among MDR Isolates Was Blactx-M-15 10 (15.6%). The Most Prevalent OXA Genes Identified Was Blaoxa-396 24 (37.5%). At Least One Aminoglycoside Gene Was Identified in All MDR Isolates. The Most Frequently Identified Aminoglycoside Gene Was Aph (3')-Iib (57.6%). Two Distinct Carbapenemase Genes Identified Were Blavim-2 And Blandm-1. Significant Mexr and Ampd Mutations Were Found Among MDR Isolates With 25 (75.8%) And 29 (87.9%) Respectively. Most Of Carbapenem-Non-Susceptible Isolates Were Exhibited Mutations in Mexr and Nalc Genes. Colistin Resistant Isolates Were Contained At Least One Mutation in Pmrb Gene. Multi-Locus Sequencing Type Analysis Revealed Two Global High-Risk Clone Types (ST235 And ST277) And Two Novel Sts (ST5135 And ST5136).
Conclusion: This Study Showed the Presence of Diverse Virulence and Resistance Determinants in P. Aeruginosa. The Finding of Genetically Related Isolates Shows Potential Clonal Spread Within Hospital. This Showed the Need for Strengthen Infection Prevention Strategies in Healthcare Settings to Monitor Spread of Resistant Strains
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Pseudomonas aeruginosa, biofilm, multidrug-resistance, serotypes, virulence genes, Ethiopia