Abstract

Background:
Pseudomonas aeruginosa is an opportunistic pathogen with the characteristics of a multidrug-resistant (MDR) antibiotic-resistant and highly virulent pathogen. Its presence in aquaculture systems poses significant threats and concerns for fish health, farm productivity, and potential zoonotic risk.

Aim:
This study aimed to assess the distribution of P. aeruginosa virulence genes, their antibiotic resistance profiles, and the genetic relationships of isolates from fish collected from major aquaculture systems in Central Iraq.

Methods:
A total of 158 fish were collected from six aquaculture sites in Central Iraq (three lake farms and three floating cage systems). Isolation was performed on cetrimide agar, and the species was identified through 16S rRNA amplification and sequencing. Antimicrobial susceptibility was tested using the disk diffusion technique for 13 antimicrobial agents in accordance with Clinical and Laboratory Standards Institute (CLSI) standards. The presence of nine virulence genes and five major antibiotic resistance genes was determined using polymerase chain reaction (PCR). To investigate clonal similarities/relations and geographic cladding, RAPD-PCR was performed.

Results:
Subsequently, 62 samples were examined and confirmed to be P. aeruginosa using 16S rRNA PCR and sequencing. Isolate phenotypic antimicrobial susceptibility testing showed 100% resistance to tetracycline and Cefuroxime / Cefuroxime sodium. Additionally, 92% of the samples were affected by MDR. Gene studies revealed that 41.9% of isolates contained all nine tested virulence factors. There were also distinct genetic groups in the RAPD-PCR analysis, possibly due to farming location. Pseudomonas aeruginosa was confirmed in 39.24% (62/158) of the samples. All isolates showed multiple antibiotic resistance indices of >0.2. The most frequently identified favorable attributes (virulence) of the pathogens were: fliC (74.19%), phz (70.96%), rhlI (61.29%), and pvd (56.45%). The formation of nine virulence patterns was recorded, with Pattern V1 (containing all genes) being the most prevalent (21%). The resistance genes aac(6′)-Ib, tetA, and blaTEM were found in 91.93%, 83.87%, and 82.25% of the isolates, respectively. RAPD-PCR revealed six geographically distinct clades that corresponded to particular fish farms, suggesting local clonal circulation and little intra-farm exchange.

Conclusion:
The results of the investigations confirm that P. aeruginosa inhabiting and genetically diverse populations in Iraqi aquaculture carry MDR and significantly virulent genotypes.

Key words: Antibiotic resistance; Aquaculture; Pseudomonas aeruginosa; Virulence genes.