SEARCHCROSS-SECTIONALveterinary medicine, animal science

cross-sectional·veterinary medicine, animal science, microbiology, epidemiology·PMC10921648

Phenotypic, molecular detection, and Antibiotic Resistance Profile (MDR and XDR) of Aeromonas hydrophila isolated from Farmed Tilapia zillii and Mugil cephalus

BMC Veterinary Research · 9 authors, 9 centres

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This study found that 31.6% of farmed Tilapia zillii and Mugil cephalus from Egypt were infected with Aeromonas hydrophila, with most isolates carrying multiple virulence genes and showing high resistance to β-lactam antibiotics. Alarmingly, 63.1% of isolates were multidrug-resistant (MDR) and 28.9% were extensively drug-resistant (XDR), including resistance to last-resort antibiotics. These findings highlight a serious public health risk from antibiotic misuse in aquaculture and the potential for resistant bacteria to enter the food chain.

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**Background** Aeromonas hydrophila is a Gram-negative bacterium that causes hemorrhagic septicemia and mass mortalities in farmed and wild fish, leading to significant economic losses in aquaculture worldwide. The pathogenicity of A. hydrophila is linked to multiple virulence genes encoding toxins and extracellular products. The widespread and often unregulated use of antibiotics in aquaculture has led to the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains, posing a risk to both fish health and human health through the food chain. This study aimed to assess the prevalence, virulence gene profile, and antibiotic resistance patterns of A. hydrophila isolated from clinically affected Tilapia zillii and Mugil cephalus in the Suez Canal region of Egypt.

**Methods** A total of 120 clinically affected fish (60 T. zillii and 60 M. cephalus) were collected from private fish farms in the Suez Canal area, Ismailia Governorate, Egypt, during different seasons (15 fish per species per season). Fish with external lesions such as hemorrhages, fin rot, distended abdomen, and skin darkening were sampled. Kidney, spleen, liver, and gills were aseptically collected and cultured on Rimler-Shotts agar and Aeromonas agar base supplemented with ampicillin. Isolates were identified phenotypically by Gram staining, motility, oxidase, catalase, O/F test, novobiocin sensitivity, hemolytic activity, proteolytic activity, and API-20 NE kit. Molecular identification was performed by PCR amplification of the 16S rRNA gene. Virulence genes (aerA, ser, alt, ast, act, hlyA, nuc, aha) and antimicrobial resistance genes (blaPSE1, blaSHV, sul1, tetA) were detected by PCR. Antimicrobial susceptibility was tested by disk diffusion against 12 antibiotics from 7 classes. The multiple antibiotic resistance (MAR) index was calculated. A pathogenicity test was conducted by intraperitoneally injecting 240 apparently healthy T. zillii with selected A. hydrophila isolates (3×10^8 CFU/mL) and monitoring mortality for 14 days.

**Key Results** - Prevalence: A. hydrophila was isolated from 38 of 120 fish (31.6%): 29 from T. zillii (48.3%) and 9 from M. cephalus (15%). The highest isolation was from liver (42.1%), followed by kidney (36.8%), spleen (13.1%), and gills (7.9%). Seasonal prevalence was highest in summer (44.73%), followed by winter (28.94%), spring (15.78%), and autumn (10.52%). - Virulence genes: All 38 isolates carried at least one virulence gene. The most prevalent was aerA (57.9%), followed by ser (28.9%), alt (26.3%), nuc (18.4%), ast (13.1%), act (7.9%), hlyA (7.9%), and aha (0%). - Antimicrobial resistance: All isolates were resistant to oxacillin and ampicillin (100%), and highly resistant to amoxicillin-clavulanic acid (89.5%), cefotaxime (89.5%), and cefadroxil (89.5%). Susceptibility was high to levofloxacin (100%), gentamicin (94.7%), ciprofloxacin (89.5%), and amikacin (86.6%). Resistance to doxycycline was 60.5%, polymyxin B 50%, and trimethoprim-sulfamethoxazole 42.1%. - Resistance genes: All isolates carried blaPSE1 (100%), 60.5% carried tetA, 42.1% carried sul1, and 42.1% carried blaSHV. - MDR/XDR: 63.1% (24/38) of isolates were MDR (resistant to ≥3 antimicrobial classes) and 28.9% (11/38) were XDR (resistant to ≥11 antimicrobial classes). The MAR index ranged from 0.16 to 0.83. - Pathogenicity test: Seven selected isolates caused 80–100% mortality in experimentally infected T. zillii. Isolates carrying the highest number of virulence and resistance genes (11/12) caused 100% mortality.

**Clinical Implications** This study demonstrates that A. hydrophila isolated from farmed fish in Egypt carry a high burden of virulence and antibiotic resistance genes, with a significant proportion being MDR or XDR. The high prevalence of resistance to β-lactams and the presence of resistance genes to tetracyclines and sulfonamides reflect the overuse of these antibiotics in aquaculture. The strong correlation between phenotypic resistance and resistance genes (e.g., doxycycline with tetA, trimethoprim-sulfamethoxazole with sul1) confirms the genetic basis of resistance. The high mortality in pathogenicity tests underscores the virulence of these strains. These findings raise serious public health concerns about the transmission of resistant bacteria and resistance genes from aquaculture to humans through fish consumption or environmental contamination. Strict regulation of antibiotic use in aquaculture, routine surveillance of resistance patterns, and development of alternative disease control strategies are urgently needed.

STUDY TYPE

cross-sectional

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veterinary medicine

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ORIGINAL PAPER

PMC10921648

Phenotypic, molecular detection, and Antibiotic Resistance Profile (MDR and XDR) of Aeromonas hydrophila isolated from Farmed Tilapia zillii and Mugil cephalus

BMC Veterinary Research

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ABSTRACT

Abstract In the present study, Aeromonas hydrophila was isolated from Tilapia zillii and Mugil cephalus samples collected during different seasons from various Suez Canal areas in Egypt. The prevalence of A. hydrophila , virulence genes, and antibiotic resistance profile of the isolates to the commonly used antibiotics in aquaculture were investigated to identify multiple drug resistance (MDR) and extensive drug-resistant (XDR) strains. In addition, a pathogenicity test was conducted using A. hydrophila , which was isolated and selected based on the prevalence of virulence and resistance genes, and morbidity of natural infected fish. The results revealed that A. hydrophila was isolated from 38 of the 120 collected fish samples (31.6%) and confirmed phenotypically and biochemically. Several virulence genes were detected in retrieved A. hydrophila isolates, including aerolysin aer A (57.9%), ser (28.9%), alt (26.3%), ast (13.1%), act (7.9%), hly A (7.9%), and nuc (18.4%). Detection of antibiotic-resistant genes revealed that all isolates were positive for bla pse 1 (100%), bla SHV (42.1%), tet A (60.5%), and sul 1 (42.1%). 63.1% of recovered isolates were considered MDR, while 28.9% of recovered isolates were considered XDR. Some isolates harbor both virulence and MDR genes; the highest percentage carried 11, followed by isolates harboring 9 virulence and resistance genes. It could be concluded that the high prevalence of A. hydrophila in aquaculture species and their diverse antibiotic resistance and virulence genes suggest the high risk of Aeromonas infection and could have important implications for aquaculture and public health. Keywords: Prevalence; Aeromonas hydrophila ; Virulence genes; Antibiotic resistance, cultured freshwater fish

CITATION EXPORT

Hala F Ayoub, Ahmed R khafagy, Aboelkair M Esawy et al.. Phenotypic, molecular detection, and Antibiotic Resistance Profile (MDR and XDR) of Aeromonas hydrophila isolated from Farmed Tilapia zillii and Mugil cephalus. BMC Veterinary Research. (2024). https://doi.org/10.1186/s12917-024-03942-y

AUTHORS

9 · 9 CENTRES

Hala F Ayoub, Ahmed R khafagy, Aboelkair M Esawy, Noura Abo El-moaty, Khairiah Mubarak Alwutayd, Abdallah Tageldein Mansour, Reham A Ibrahim, Dalia A Abdel-moneam, Reham M El-Tarabili

1Department of Fish Health and Management, Central Laboratory for Aquaculture Research (CLAR), Agricultural Research Center, Abo-Hammad, Sharqia, Abbassa 44662 Egypt
2Department of Bacteriology, Immunology, and Mycology, Faculty of Veterinary Medicine, Ain Shams University, Cairo, Egypt
3Department of Microbiology, Animal Health Research Institute, Mansoura branch, Mansoura, Egypt
4Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh, 11671 Saudi Arabia
5Fish and Animal Production and Aquaculture Department, College of Agriculture and Food Sciences, King Faisal University, P.O. Box 420, Al-Ahsa, 31982 Saudi Arabia
6Fish and Animal Production Department, Faculty of Agriculture (Saba Basha), Alexandria University, Alexandria, 21531 Egypt
7Microbiology Department, National Institute of Oceanography and Fisheries (NIOF), Cairo, Egypt
8Department of Aquatic Animal Medicine and Management, Faculty of Veterinary Medicine, Cairo University, Giza, 12211 Egypt
9Department of Bacteriology, Immunology and Mycology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, 41522 Egypt