PO03 | A NOVEL HALOBACTERIOVORAX AS A BIOCONTROL AGENT AGAINST ANTIMICROBIAL-RESISTANT ESCHERICHIA COLI ON FOOD-CONTACT SURFACES

Stefania Di Lullo, Silvia Pieralisi, Gabriele Angelico, Diego Maiolatesi, Giulia Talevi, Sara Nardi, Francesca Leoni, Elena Rocchegiani, Donatella Ottaviani | Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati”, Ancona, Italy.

Objective. The study aims to evaluate the predatory potential of Halobacteriovorax (HE7) belonging to the group of predatory bacteria known as Bdellovibrio and like organisms (BALOs) against antimicrobial-resistant (AMR) Escherichia coli on food-contact surfaces under simulated experimental conditions that replicate food processing environments.

Methods. A strain of Halobacteriovorax (HE7) was isolated from an estuarine environment and identified by 16S rRNA analysis and Sanger sequencing, using a multidrug-resistant E. coli strain (E7) as prey, carrying the blaCTX-M-14 and blaCMY-2 genes encoding ESBL and AmpC β-lactamases, respectively, with broad β-lactam resistance. Challenge tests were conducted using different predator/prey ratios on laminated surfaces (900 cm²), divided into 100 cm² sections and identified as Test Surface 1 (HE7+E7), Prey Control Surface 2 (E7), and Predator Control Surface 3 (HE7). The surfaces were disinfected, rinsed, dried, and treated with a sterile neutralizing solution before contamination. A ratio of 10⁶ PFU/10³ CFU was used, selected based on our previous studies. The surfaces were inoculated, allowed to dry for 30 minutes, and sampled to assess bacterial adhesion; they were then inoculated with HE7 for the test and with sterile BALOs enrichment medium for the controls. After two hours, the surfaces were sampled again; sterile sponges were used at both time points. The resulting suspensions were subjected to serial dilutions and analyzed by plating on MacConkey agar for prey titration and by plaque assay for predator determination.

Results. Two hours after spreading HE7 over surface 1, which had previously been contaminated with E7, an approximately 3-log reduction in the E. coli load was observed, with counts decreasing from 1.5 × 10³ CFU/100 cm² to below the limit of detection (<10 CFU/100 cm²). This reduction was not observed in the controls without the predator (surface 2), where the concentration of E7 remained essentially stable compared to the initial time, confirming the absence of effects related to experimental conditions. Concomitantly, HE7 demonstrated persistence on surfaces. In controls without prey (surface 3), a slight reduction in the predator was observed, whereas in the presence of E. coli, its stability was higher, supporting its predatory activity.

Conclusions. The results indicate a rapid and substantial reduction in AMR E. coli contamination on food-contact surfaces mediated by Halobacteriovorax, confirming its potential use as a biological control agent and supporting the use of predatory bacteria as a complementary and sustainable strategy in the management of antimicrobial resistance, with a possible reduction in the selective pressure associated with conventional treatments. The observed predatory efficacy and persistence of Halobacteriovorax in the presence of prey support the potential of predatory bacteria to maintain their activity on food-contact surfaces even under simulated environmental conditions. This highlights the potential of Halobacteriovorax and, more generally, of BALOs as an innovative tool for controlling microbial contamination on food-contact surfaces, with important implications for food safety and health. Further studies will be needed to evaluate its safety of use, stability, and efficacy under real-world operating conditions, as well as its applicability in environmental and food contexts.
These works were financed by the Ministero della Salute (RC04/2025 and (RC07/2023).