Emerging MDR-Pseudomonas aeruginosa in fish commonly harbor oprL and toxA virulence genes and blaTEM, blaCTX-M, and tetA antibiotic-resistance genes

Abstract

This study aimed to investigate the prevalence, antibiogram of Pseudomonas aeruginosa (P. aeruginosa), and the distribution of virulence genes (oprL, exoS, phzM, and toxA) and the antibiotic-resistance genes (bla_TEM, tetA, and bla_CTX-M). A total of 285 fish (165 Oreochromis niloticus and 120 Clarias gariepinus) were collected randomly from private fish farms in Ismailia Governorate, Egypt. The collected specimens were examined bacteriologically. P. aeruginosa was isolated from 90 examined fish (31.57%), and the liver was the most prominent infected organ. The antibiogram of the isolated strains was determined using a disc diffusion method, where the tested strains exhibited multi-drug resistance (MDR) to amoxicillin, cefotaxime, tetracycline, and gentamicin. The PCR results revealed that all the examined strains harbored (oprL and toxA) virulence genes, while only 22.2% were positive for the phzM gene. On the contrary, none of the tested strains were positive for the exoS gene. Concerning the distribution of the antibiotic resistance genes, the examined strains harbored bla_TEM, bla_CTX-M, and tetA genes with a total prevalence of 83.3%, 77.7%, and 75.6%, respectively. Experimentally infected fish with P. aeruginosa displayed high mortalities in direct proportion to the encoded virulence genes and showed similar signs of septicemia found in the naturally infected one. In conclusion, P. aeruginosa is a major pathogen of O. niloticus and C. gariepinus. oprL and toxA genes are the most predominant virulence genes associated with P. aeruginosa infection. The bla_CTX-M, bla_TEM, and tetA genes are the main antibiotic-resistance genes that induce resistance patterns to cefotaxime, amoxicillin, and tetracycline, highlighting MDR P. aeruginosa strains of potential public health concern.

Figure 1

Clinical examination of naturally infected fish (a) catfish (Clarias gariepinus) showing irregular hemorrhages on external body surfaces, especially at the ventral part of the abdomen and around the vent (white arrows), (b) Nile tilapia (Oreochromis niloticus) showing scattered hemorrhagic spots (white arrows), detached scales (*), and fins erosion (black arrows).

Figure 2

Post-mortem examination of naturally infected fish (a) Nile tilapia (Oreochromis niloticus) showing pale liver (black arrow), necrotic gills (white arrow), engorged spleen (*), serous fluid exudate, and congested kidney (#), (b) catfish (Clarias gariepinus) showing friable liver (#), congested kidneys (white arrows), and engorged spleen (*).

Figure 3

Electrophoretic pattern of primers targeting oprL gene. Lane L: 100 bp molecular weight ladder (Fermentas, Thermo Scientific, Germany); lane Pos: the positive control (Reference strain from Animal Health Research Institute in Dokki, Cairo, Egypt), and lane Neg: the negative control (template without DNA). Lanes 1–7: the specific DNA product (504 bp) amplified from representative isolates of P. aeruginosa.

Figure 4

Electrophoretic pattern of primers targeting toxA gene. Lane L: 100 bp molecular weight ladder (Fermentas, Thermo Scientific, Germany); lane Pos: the positive control (Reference strain from Animal Health Research Institute in Dokki, Cairo, Egypt), and lane Neg: the negative control (template without DNA). Lanes 1–7: the specific DNA product (396 bp) amplified from representative isolates of P. aeruginosa.

Figure 5

Electrophoretic pattern of primers targeting phzM gene. Lane L: 100 bp molecular weight ladder (Fermentas, Thermo Scientific, Germany); lane Pos: the positive control (Reference strain from Animal Health Research Institute in Dokki, Cairo, Egypt), and lane Neg: the negative control (template without DNA). Lanes 1–7: the specific DNA product (875 bp) amplified from representative strains of P. aeruginosa.

Figure 6

Electrophoretic pattern of primers targeting exoS gene. Lane L: 100 bp molecular weight ladder (Fermentas, Thermo Scientific, Germany); lane Pos: the positive control (Reference strain from Animal Health Research Institute in Dokki, Cairo, Egypt), and lane Neg: the negative control (template without DNA). Lanes 1–7: the specific DNA product (118 bp) amplified from representative strains of P. aeruginosa.

Figure 7

Electrophoretic pattern of primers targeting bla_TEM gene. Lane L: 100 bp molecular weight ladder (Fermentas, Thermo Scientific, Germany); lane Pos: the positive control (Reference strain from Animal Health Research Institute in Dokki, Cairo, Egypt), and lane Neg: the negative control (template without DNA). Lanes 1–7: the specific DNA product (516 bp) amplified from representative isolates of P. aeruginosa.

Figure 8

Electrophoretic pattern of primers targeting tetA gene. Lane L: 100 bp molecular weight ladder (Fermentas, Thermo Scientific, Germany); lane Pos: the positive control (Reference strain from Animal Health Research Institute in Dokki, Cairo, Egypt), and lane Neg: the negative control (template without DNA). Lanes 1–7: the specific DNA product (576 bp) amplified from representative strains of P. aeruginosa.

Figure 9

Electrophoretic pattern of primers targeting bla_CTM gene. Lane L: 100 bp molecular weight ladder (Fermentas, Thermo Scientific, Germany); lane Pos: the positive control (Reference strain from Animal Health Research Institute in Dokki, Cairo, Egypt), and lane Neg: the negative control (template without DNA). Lanes 1–5: the specific DNA product (593 bp) amplified from representative strains of P. aeruginosa.

Figure 10

Cumulative mortality of O. niloticus subjected to intraperitoneal injection with 0.1 ml of virulent P. aeruginosa strain A (T1) and P. aeruginosa strain B (T2) at concentration of 3 × 10⁷ cells mL^-1.