Refining the gut colonization Zophobas morio larvae model using an oral administration of multidrug-resistant Escherichia coli.

Background: The darkling beetle Zophobas morio can be implemented as an alternative in vivo model to study different intestinal colonization aspects. Recently, we showed that its larvae can be colonized by multidrug-resistant Escherichia coli strains administered via contaminated food (for 7 days) for a total experimental duration of 28 days.

Method: In the present work, we aimed to shorten the model to 14 days (T14) by administering the previously used CTX-M-15 ESBL-producing ST131 Escherichia coli strain Ec-4901.28 via a single oral administration (5 µL dose of 10⁸ CFU/mL) , using a blunt 26s-gauge needle connected to a 250 μL gastight syringe. Force-feeding was performed either without or with (larvae placed on ice for 10 minutes before injection) anesthesia. In addition, phage-treated larvae were orally injected with 10 µL of INTESTI bacteriophage cocktail (∼10^5-6 PFU/mL) on days 4 (T4) and 7 (T7) .

Results: Growth curve analyses showed that, while larvae rapidly became colonized with Ec-4901.28 (T1, ∼10^6-7 CFU/mL) , only those anesthetized maintained a high bacterial load (∼10^2-3vs. ∼10^5-6 CFU/mL) and survival rate (76% vs. 99%; P<0.001) by T14. Moreover, bacteriophage administration to anesthetized larvae significantly reduced the bacterial count of INTESTI-susceptible Ec-4901.28 at T14 (5.17 × 10⁵vs. 2.26 × 10^4, for non-treated and phage-treated larvae, respectively; P=0.04) .

Conclusion: The methodological refinements applied to establish the intestinal colonization model simplify the use of Z. morio larvae, facilitate prompt evaluation of novel decolonization approaches and reduce experiments involving vertebrate animals in accordance with the Replacement, Reduction and Refinement principles.