Bacillus velezensis AP183 Inhibits Staphylococcus aureus Biofilm Formation and Proliferation in Murine and Bovine Disease Models.

Andrew D Brannen,Abdulaziz Al Mouslem,Chengming Wang,Herris Maxwell,Edward W Davis,Peter Panizzi,Mary J Sandage,Mark R Liles,Shamima Nasrin,Cody R Rasmussen-Ivey,Sayma Afroj,Terri Hathcock

doi:10.3389/fmicb.2021.746410

Abstract

The increasing frequency of S. aureus antimicrobial resistance has spurred interest in identifying alternative therapeutants. We investigated the S. aureus-inhibitory capacity of B. velezensis strains in mouse and bovine models. Among multiple B. velezensis strains that inhibited S. aureus growth in vitro, B. velezensis AP183 provided the most potent inhibition of S. aureus proliferation and bioluminescence in a mouse cutaneous wound (P = 0.02). Histology revealed abundant Gram-positive cocci in control wounds that were reduced in B. velezensis AP183-treated tissues. Experiments were then conducted to evaluate the ability of B. velezensis AP183 to prevent S. aureus biofilm formation on a tracheostomy tube substrate. B. velezensis AP183 could form a biofilm on a tracheostomy tube inner cannula substrate, and that this biofilm was antagonistic to S. aureus colonization. B. velezensis AP183 was also observed to inhibit the growth of S. aureus isolates originated from bovine mastitis cases. To evaluate the inflammatory response of mammary tissue to intramammary inoculation with B. velezensis AP183, we used high dose and low dose inocula in dairy cows. At the high dose, a significant increase in somatic cell count (SCC) and clinical mastitis was observed at all post-inoculation time points (P < 0.01), which resolved quickly compared to S. aureus-induced mastitis; in contrast, the lower dose of B. velezensis AP183 resulted in a slight increase of SCC and no clinical mastitis. In a subsequent experiment, all mammary quarters in four cows were induced to have grade 1 clinical mastitis by intramammary inoculation of a S. aureus mastitis isolate; following mastitis induction, eight quarters were treated with B. velezensis AP183 and milk samples were collected from pretreatment and post-treatment samples for 9 days. In groups treated with B. velezensis AP183, SCC and abundance of S. aureus decreased with significant reductions in S. aureus after 3 days post-inoculation with AP183 (P = 0.04). A milk microbiome analysis revealed significant reductions in S. aureus relative abundance in the AP183-treated group by 8 days post-inoculation (P = 0.02). These data indicate that B. velezensis AP183 can inhibit S. aureus biofilm formation and its proliferation in murine and bovine disease models.

Highlights

S. aureus is commonly responsible for cutaneous infections in human and veterinary medicine, with the rising prevalence of antibiotic resistant strains prompting the need for novel strategies to combat infection (Guo et al, 2020)
The final concentrations of S. aureus Xen29 and B. velezensis spores per wound were approximately 1.0 × 107 CFU respectively. The results in these small number of animals indicated that co-administration of S. aureus Xen29 with spores of B. velezensis AP183 resulted in the greatest inhibition of S. aureus Xen29 growth and bioluminescence as compared to the other B. velezensis strains AP191, AP218, AB01, or AP143, with B. velezensis AP191 showing a moderate degree of S. aureus inhibition
The in vivo efficacy observed for B. velezensis AP183 in inhibiting S. aureus in a mouse wound model, and the strong in vitro growth inhibitory activity observed for B. velezensis against all the mastitis-derived S. aureus isolates tested in this study suggested that B. velezensis AP183 may have the ability to inhibit a broad spectrum of Gram-positive bacterial taxa that can induce mastitis

Summary

Introduction

S. aureus is commonly responsible for cutaneous infections in human and veterinary medicine, with the rising prevalence of antibiotic resistant strains prompting the need for novel strategies to combat infection (Guo et al, 2020). Asymptomatic carriage of S. aureus in humans is estimated to be approximately 20 percent, though the pathogen is common to a variety of acute and chronic skin pathologies including impetigo, cellulitis, furuncles, scalded skin syndrome, and mastitis (Iwatsuki et al, 2006; Delgado et al, 2011). Cutaneous infections are burdensome in agriculture as well, with bovine mastitis alone resulting in an estimated annual cost exceeding $1.7 billion to the dairy industry and S. aureus as the causative pathogen in 20% of cases (Wells and Ott, 1998; Shim et al, 2004). The primary causative agents of IMI are bacterial, including Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus uberis, and Escherichia coli (Barkema et al, 2009; Piessens et al, 2011; Supre et al, 2011). Antibiotic use remains the most common treatment against mastitis (Barlow, 2011); the use of antibiotics may foster bacterial antimicrobial resistance, but can cause contamination of milk and meat, potentially contributing to dissemination of antibiotic resistance in the environment and in humans (Oliver and Murinda, 2012; Sharma et al, 2017)

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