A Multidrug-Resistant Escherichia coli Caused the Death of the Chinese Soft-Shelled Turtle (Pelodiscus sinensis)

Abstract

The rapid increase in drug resistance in recent years has become a significant global public health concern. Escherichia coli are ubiquitous bacteria, widely distributed in various environments. This study isolated a bacterial strain (HD-593) from diseased Chinese soft-shelled turtles (Pelodiscus sinensis). The bacterium was identified based on morphology, biochemical tests, and 16S rRNA sequencing, confirming it as E. coli. Drug susceptibility tests revealed that the HD-593 strain was highly resistant to ceftriaxone, enrofloxacin, doxycycline, sulfadiazine, gentamicin, neomycin, florfenicol, carbenicillin, cefradine, erythromycin, penicillin, ampicillin, midecamycin, and streptomycin. Resistance gene analysis confirmed the presence of quinolone resistance genes (oqxA and oqxB), aminoglycoside resistance genes (aac(3)-II and aphA1), a β-lactam resistance gene (blaTEM), and an acylaminol resistance gene (floR) in HD-593. The median lethal dose (LD50) of HD-593 for P. sinensis was 6.53 × 10⁵ CFU/g. Biochemical analysis of serum revealed that HD-593 infection caused a significant reduction in total protein, albumin, and globulin levels, while markedly increasing the levels of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase. Histopathological analysis revealed severe intestinal damage characterized by villi detachment and muscle cell necrosis. Additionally, extensive splenocyte necrosis with nuclear marginalization, glomerular swelling, and pronounced hepatic steatosis accompanied by distended sinusoids were observed. This study identified a multidrug-resistant E. coli strain from deceased P. sinensis, suggesting that drug resistance genes may circulate in aquaculture ecosystems, posing potential risks to aquaculture.

Keywords: Escherichia coli; Pelodiscus sinensis; drug resistance genes; multidrug-resistant.

Figure 1

Presentation of the diseased P. sinensis. (A): Boils on the backside (red arrows), (B): boils on the abdomen (red arrows), (C): anatomy of sick turtle, (D): enlargement of the liver, (E): no food and bleeding in the intestines.

Figure 2

Morphological features of HD-593 bacteria. (A): HD-593 bacteria growing in BHI solid medium. (B): HD-593 bacteria Gram stain (scale bar: 10 μm). (C): The scanning electronic micrograph of the HD-593 (scale bar: 3 μm).

Figure 3

The phylogenetic tree of the HD-593 (red triangle) isolate based on the neighbor-joining technique in MEGA 11. Each branch is annotated with a percentage, indicating the bootstrap values based on 1000 replicates. The scale bars represent the number of substitutions per locus.

Figure 4

Agarose gel electrophoresis of PCR products of the drug resistance genes. M: DL1000 marker, 1: blaTEM, 2: oqxA, 3: aac(3)-II, 4: mcr-4, 5: floR, 6: aphA1, 7: ereA, 8: oqxB. Original image—Figure S2.

Figure 5

Cumulative mortality of P. sinensis infected with E. coli HD-593 at different bacterial doses (1 × 10⁴–1 × 10⁸ CFU/g body weight). Groups (n = 30/group) were intraperitoneally injected with bacterial suspensions or PBS (control). Mortality was monitored for 10 days. Mortality reached 100% at the highest dose (1 × 10⁸ CFU/g). The LD₅₀ value (6.53 × 10⁵ CFU/g) was calculated using the Reed–Muench method.

Figure 6

Serum biochemical parameters in healthy (H) and diseased (D) P. sinensis. Levels of total protein (TP), albumin (ALB), globulin (GLB), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and alanine aminotransferase (ALT) were quantified. Values are mean ± SD (* p < 0.05; ** p < 0.01).