In Vitro Evaluation of Phytobiotic Mixture Antibacterial Potential against Enterococcus spp. Strains Isolated from Broiler Chicken

Abstract

Enterococcus spp. are normal intestinal tract microflorae found in poultry. However, the last decades have shown that several species, e.g., Enterococcus cecorum, have become emerging pathogens in broilers and may cause numerous losses in flocks. In this study, two combinations (H1 and H2) of menthol, 1,8-cineol, linalool, methyl salicylate, γ-terpinene, p-cymene, trans-anethole, terpinen-4-ol and thymol were used in an in vitro model, analyzing its effectiveness against the strains E. cecorum, E. faecalis, E. faecium, E. hirae and E. gallinarum isolated from broiler chickens from industrial farms. To identify the isolated strains classical microbiological methods and VITEK 2 GP cards were used. Moreover for E. cecorum a PCR test was used.. Antibiotic sensitivity (MIC) tests were performed for all the strains. For the composition H1, the effective dilution for E. cecorum and E. hirae strains was 1:512, and for E. faecalis, E. faecium and E. gallinarum, 1:1024. The second mixture (H2) showed very similar results with an effectiveness at 1:512 for E. cecorum and E. hirae and 1:1024 for E. faecalis, E. faecium and E. gallinarum. The presented results suggest that the proposed composition is effective against selected strains of Enterococcus in an in vitro model, and its effect is comparable to classical antibiotics used to treat this pathogen in poultry. This may suggest that this product may also be effective in vivo and provide effective support in the management of enterococcosis in broiler chickens.

Keywords: E. cecorum; Enterococcus spp.; broiler; osteomyelitis; phytobiotics; resistance genes.

Figure 1

Percentage of susceptible, intermediate and resistant isolates of (A) E. cecorum (n = 154), (B) E. faecalis (n = 453), (C) E. gallianrum (n = 57) and (D) E. hirae (n = 29). AMX—Amoxicillin, AMX/CL—Amoxicillin–clavulanic acid, DOX—Doxycycline, ERY—Erythromycin, NOR—Norfloxacin; R—resistant, I—intermediate, S—susceptible.

Figure 2

The most frequently observed post-mortem lesions associated with E. cecorum infection in broiler chickens are related to necrotic changes in the femoral head. Clinically, it manifests itself as unilateral and/or bilateral lameness. The lack of movement and, consequently, the lack of water and feed intake results in lower weight gain, worse overall health status and decreased uniformity of the flock.

Figure 3

During post-mortem evaluation at the slaughterhouse, a large percentage of flocks with clinical symptoms of E. cecorum infection, despite treatment, showed a number of changes in the carcass that reduced its quality. A large group of changes was related to the inability to move properly (lameness), being trapped and trampled by other birds (bedsores, hematomas, abscesses, skin scratches, etc.).

Figure 4

A common post-mortem observation was the large variation in flock weights (low uniformity) that accompanied birds after E. cecorum infection after the 20th day of life.

Figure 5

Schematic illustration of the steps carried out in this study.