Evaluation of Single and Multi-Strain Probiotics with Gentamicin Against E. coli O157:H7: Insights from In Vitro and In Vivo Studies

Abstract

The emergence of antibiotic-resistant food-borne pathogens, especially Escherichia coli O157:H7, highlights the urgent need for innovative treatment strategies, particularly in light of rising resistances and the ongoing controversy surrounding antibiotic use in response to E. coli O157:H7 infections. To address this issue, we explored the potential of single- and multi-strain probiotics, both independently and in combination with gentamicin, through a series of in vitro and in vivo experiments. In vitro, gentamicin alone produced a mean inhibition zone of 12.9 ± 2.27 mm against E. coli O157:H7. The combination of gentamicin with single-strain probiotics (P1) increased the inhibition zone to 16.5 ± 2.24 mm (p < 0.05), while the combination with multi-strain probiotics (P2) resulted in the largest inhibition zone of 19 ± 2.8 mm (p < 0.05). In vivo, mice infected with E. coli O157:H7 and treated with P2, gentamicin (G), or their combination (G+P2), achieved 100% survival, no pathological symptoms, and full weight recovery within seven days. Conversely, mice treated with P1 or G+P1 exhibited lower survival rates (71.4% and 85%, respectively) and slower weight recovery. Hematological parameters improved across all groups, but kidney function analysis showed significantly higher serum creatinine levels in the P1, G, G+P1, and G+P2 groups compared to the P2 group (P1: 0.63 ± 0.15 mg/dL; G: 0.34 ± 0.09 mg/dL; G+P1: 0.53 ± 0.19 mg/dL; G+P2: 0.5 ± 0.23 mg/dL vs. P2: 0.24 ± 0.2 mg/dL). Histological analysis showed better intestinal and kidney tissue recovery in the P2 group, while the P1 and G+P1 groups exhibited abnormal ileal structures and severe cortical bleeding. These findings highlight the promise of multi-strain probiotics, alone or in conjunction with antibiotics, as a therapeutic strategy for E. coli O157:H7 infections. However, the nephrotoxicity associated with gentamicin co-administration remains a limitation, warranting further studies to optimize this approach.

Keywords: E. coli O157:H7; gentamicin; infection; probiotics.

Figure 1

Agarose gel electrophoresis of DNA fragments generated by PCR for fliC gene (A) and rfbE gene (B) of E. coli O157:H7 isolates. In (A), Lane 8: DNA molecular weight marker (100 bp); Lane 11: Negative control; Lanes 2, 3, 5, 6, 7, 9, 10, 12, 13, 14, 15: E. coli O:157:H7 isolates. In (B), Lane 10: DNA molecular weight marker (100 bp); Lane 7: Negative control; Lanes 2, 3, 4, 5, 9: E. coli O:157:H7 isolates.

Figure 2

The antimicrobial effect of single and multi-strained probiotics alone and in combination with gentamicin against E. coli O157:H7 isolates. Values represent the mean inhibition zone (in millimeters) ± standard deviation, calculated from multiple replicates for each treatment. * The p-value of the mean diameter of the zone of inhibition of different groups was <0.05 using one-way ANOVA test followed by Tukey’s test, compared to gentamicin.

Figure 3

A picture of infected mice with bruising and fur abnormalities.

Figure 4

Histopathology in E. coli O157:H7-infected mice. Normal intestinal histology was observed in the control group. Infected mice exhibited atrophic villi and intraglandular bleeding (indicated by the arrow).

Figure 5

Histopathology in E. coli O157:H7-infected mice. Normal intestinal histology was observed in the P2 group. Mice treated with P1 displayed intraglandular bleeding (highlighted by circle).

Figure 6

Histopathology in E. coli O157:H7-infected mice. Normal intestinal histology was observed in the gentamicin group and the G+P2 combination group. Mice in the G+P1 combination group exhibited mild intraglandular bleeding (highlighted by the circle).

Figure 6

Histopathology in E. coli O157:H7-infected mice. Normal intestinal histology was observed in the gentamicin group and the G+P2 combination group. Mice in the G+P1 combination group exhibited mild intraglandular bleeding (highlighted by the circle).

Figure 7

Renal histopathology in E. coli O157:H7-infected mice. Preservation of normal cortex in the control and P2 treatment group.

Figure 8

Renal histopathological changes in E. coli O157:H7-infected mice without treatment. Panel (A) shows bleeding in the cortical region, Panel (B) illustrates bleeding in the collecting duct, Panel (C) highlights congested blood vessels in the collecting duct, and Panel (D) displays bleeding in the calyceal system.

Figure 9

Renal histopathological changes in E. coli O157:H7-infected mice. Bleeding in the cortex was observed in all treatment groups, with the most severe bleeding in the P1 group and the least seen in mice treated with the gentamicin+P2 combination.