Growth inhibitory effect of a triple-strain bacillus-based probiotic on enterococcus cecorum isolated from broilers: an in vitro study

Enterococcus cecorum, a gram-positive coccus, plays an integral role in the composition of the gastrointestinal microbiota across a diverse array of mammalian and avian species. It is worth noting that this range encompasses not only a variety of avifauna, such as chickens, ducks, and pigeons, but also several mammalian species, including pigs, calves, horses, cats, and dogs (Baele et al., 2022). E. cecorum has been implicated sporadically in a range of human illnesses, including peritonitis, septicemia, and empyema, typically presenting in individuals with preexisting conditions or vulnerabilities such as alcohol abuse, liver cirrhosis, malnutrition, or undergoing continuous ambulatory peritoneal dialysis (De Baere et al., 2000). Human disease associated with E. cecorum has been documented in only a small number of publications (De Baere et al., 2000) (Greub et al., 1997) (Hsueh et al., 2000) (Woo et al., 2004). In the last 15 years, E. cecorum has surfaced as a noteworthy opportunistic pathogen affecting poultry. The infection attributed to E. cecorum was detected in 2002 in broilers aged 4-5 weeks, originating from Scotland (Wood et al., 2002). E. cecorum is usually recognized as a harmless commensal within the gut of broiler chickens. However, the occurrence of dysbiosis, an imbalance in the gut microbiome has the potential to compromise the gut barrier integrity resulting in a "leaky gut". This condition can facilitate intestinal bacterial overgrowth, providing an opportunity for E. cecorum to exploit weakened tight junctions with potential to translocate into the blood stream followed by transport to specific sites (e.g., joints) or organs (e.g., heart). E. cecorum has been implicated in causing bone lesions, notably chondronecrosis and osteomyelitis in broiler chickens. Disease outbreaks associated with E. cecorum were characterized by a range of symptoms including sepsis, pericarditis, local myositis, and particularly bone and joint lesions, which encompass purulent arthritis of the hock, femoral head necrosis, and T6 vertebral osteomyelitis. E. cecorum presently poses a significant threat to the global profitability of broiler operations with increased mortality rates, poor production, and increased condemnation (Dolka et al.,2017). Therefore, for poultry production, evaluation of probiotics has been undertaken to determine the potential effect of growth inhibition of E. cecorum.

From USA and West European farms, twenty-two isolates of E. cecorum were collected from broiler chickens displaying clinical signs of lameness. The goal was to evaluate the in vitro inhibitory capability of a triple-strain Bacillus-based probiotic, consisting of B. subtilis (DSM 32325), B. subtilis (DSM 32324), and B. amyloliquefaciens (DSM 25840). Out of the 22 isolates, 10 were provided by the University of Arkansas (Prof. Douglas Rhoads and Dr. Adnan Ali Khalaf Alrubaye), while the remaining 12 West-European isolates were obtained from a laboratory in France. These E. cecorum strains were isolated from various body sites including the heart, liver and joints or via cloacal swabs. 

A pathogen inhibition assay was conducted to determine the ability of the probiotic supernatant to inhibit the growth of the different E. cecorum isolates. The probiotic was cultured in BHI broth and the E. cecorum isolates were cultured in TSA-sheep blood agar at 37˚C for 24 hours. The probiotic broths were centrifugated for bacterial cell sedimentation, separating the supernatant from the precipitates through filtration. A 100 µl aliquot of this cellfree probiotic supernatant, which has previously been shown to contain active substances including bacteriocins, was subsequently combined with 100 µl of new BHI broth and 20 µl of E. cecorum in saline diluent inoculated in wells. Post-inoculum incubation to assess the inhibitory effect of the supernatant against bacterial growth was evaluated using the Optical/OD plate reader for 96 well plates by measuring the turbidity (Figure 1). All experiments were conducted in triplicate. The average OD of triplicates are calculated for both probiotic and control growth. This is the inhibition percentage for each timepoint. To calculate the percentage decrease in growth, an average of these percentage inhibition were performed to get one number per strain.

As presented in Figure 2, results revealed that the triple-strain probiotic had a very high inhibition of the growth of E. cecorum ranging from 80% and 96% for the 12 West European and 10 US isolates. 

This strong in vitro capability for direct inhibition of clinical E. cecorum isolates can be explained by the ability of the different Bacillus strains in the probiotic to release specific inhibitory substances (such as lipopeptides like surfactins and fengycins) to its surrounding that inhibit bacteria through the formation of pores in cell membranes. The in vitro activity shown in this study suggests that the probiotic should have in vivo efficacy.

In conclusion, the observed direct pathogen inhibitory effect in vitro as demonstrated through supernatant assay is just one facet of the multifaceted modes of action attributable to Bacillus-based probiotics. The action of triple-strain probiotic has been shown to reduce the in vitro growth of E. cecorum.