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Bacillus Subtilis Revives Conventional Antibiotics Against Staphylococcus

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Bacillus Subtilis Revives Conventional Antibiotics Against Staphylococcus Zhang et al. Microb Cell Fact (2021) 20:102 https://doi.org/10.1186/s12934-021-01592-5 Microbial Cell Factories RESEARCH Open Access Bacillus subtilis revives conventional antibiotics against Staphylococcus aureus osteomyelitis Fan Zhang1,2, Bowei Wang1, Shiluan Liu1,2, Yuhui Chen1,2, Yihuang Lin1,2, Zixian Liu1,2, Xianrong Zhang1,2* and Bin Yu1,2* Abstract As treatment of Staphylococcus aureus (S. aureus) osteomyelitis is often hindered by the development of antibiotic tol- erance, novel antibacterial therapeutics are required. Here we found that the cell-free supernatant of Bacillus subtilis (B. subtilis CFS) killed planktonic and bioflm S. aureus, and increased S. aureus susceptibility to penicillin and gentamicin as well. Further study showed that B. subtilis CFS suppressed the expression of the genes involved in adhesive mol- ecules (Cna and ClfA), virulence factor Hla, quorum sensing (argA, argB and RNAIII) and bioflm formation (Ica and sarA) in S. aureus. Additionally, our data showed that B. subtilis CFS changed the membrane components and increased membrane permeabilization of S. aureus. Finally, we demonstrated that B. subtilis CFS increased considerably the sus- ceptibility of S. aureus to penicillin and efectively reduced S. aureus burdens in a mouse model of implant-associated osteomyelitis. These fndings support that B. subtilis CFS may be a potential resistance-modifying agent for β-lactam antibiotics against S. aureus. Keywords: Staphylococcus aureus, Osteomyelitis, Antibiotic tolerance, Bacillus subtilis, Membrane permeabilization, Bioflm Introduction immunomodulatory proteins, toxins and superantigens, Gram-positive Staphylococcus aureus (S. aureus) has leading to death of innate immune cells and disturbance been identifed as the most common causative patho- of complement activation [5]. As the infection persists gen for osteomyelitis and other various musculoskeletal and becomes chronic, S. aureus may adhere to implanted infections [1, 2]. S. aureus osteomyelitis remains a signif- devices, lacunae-canaliculi in cortical bone or seques- cant healthcare problem in China and around the world tra, thereby forming bioflm phenotype [6, 7]. Once a due to high rates of recurrence and treatment failure [3, bioflm forms, S. aureus is 10 − 1,000 times more resist- 4]. Treatment of S. aureus infection in bone is compli- ant to antimicrobial agents than planktonic bacteria [8] cated by its vast immune evasion, persistence mecha- and induces phagocytosis dysfunction of macrophages nisms and intrinsic antibiotic resistance mechanism. S. [9]. Additionally, intracellular persistence of S. aureus aureus may secrete multiple virulence factors including in osteoblasts, macrophages, osteoclasts or osteocytes may induce immune cell evasion and antibiotic toler- *Correspondence: [email protected]; [email protected] ance of S. aureus during infection [10, 11]. Furthermore, †Fan Zhang and Bowei Wang contributed equally to this work S. aureus has such intrinsic mechanism for antibiotic 1 Division of Orthopaedics and Traumatology, Department resistance as decreasing permeability of outer membrane, of Orthopaedics, Nanfang Hospital, Southern Medical University, No.1838 North of Guangzhou Avenue, Guangzhou 510515, Guangdong, China activating drug efux systems, and producing excessive Full list of author information is available at the end of the article β-Lactamase [12–14]. © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhang et al. Microb Cell Fact (2021) 20:102 Page 2 of 15 Surgical debridement of necrotic bone combined with adjusted to an optical density at 600 nm (OD600) of 0.5 long-term administration of antibiotics is a traditional measured using a microplate spectrophotometer (CLAR- therapy to treat chronic osteomyelitis [15]. Several anti- IOstar, BMG LABTECH, Germany), approximately equal 8 biotics are used for management of S. aureus osteomy- to 1 × ­10 colony forming unit per ml (CFU/ml). elitis, such as vancomycin, tobramycin, daptomycin and clindamycin, but the rapid acquisition of resistance to Preparation of cell‑free supernatant from B. subtilis culture antibiotics by S. aureus is a signifcant problem [16–19]. and treatments Terefore, it is urgent to fnd a more efective antibac- To prepare B. subtilis CFS, B. subtilis strains were cul- terial strategy to prevent occurrence and recurrence of tured at 37℃ under shaking at 200 rpm overnight until bone infections. the cultures reached an OD 600 of 0.4 ± 0.05. Te CFS of Recently, probiotics such as Bacillus subtilis (B. sub- bacterial culture was collected by centrifugation at 6000 g tilis) has been used to prevent infection, because it is a for 10 min, and then fltered through a 0.22 μm steriliz- nonpathogenic Gram-positive bacterium which can ing-grade flter (Millipore, SLGV033RB, USA) to remove ℃ efectively maintain a benefcial microfora balance in the bacteria. Te CFS was aliquoted and stored at − 20 gastrointestinal tract of a mammalian host [20]. Accu- until the day of experimentation. mulating evidence from animal and in vitro studies sug- To evaluate the efect of B. subtilis CFS on S. aureus gests that B. subtilis produces various substances, such as genes expression, overnight culture of S. aureus strains sufactins, iturins and fengycins, which may beneft anti- was collected by a centrifuge, washed with PBS, re-sus- 8 bacterial, anti-infammatory and immunomodulatory pended at 1 × ­10 CFU/ml in TSB/PBS (1:1 v/v, control) applications [21, 22]. Specifcally, a recent report showed or TSB/B. subtilis CFS (1:1 v/v) and incubated in 6-well- that the secreted substance from B. subtilis abolished plate at 37℃ for 3 h. Finally, bacteria were collected for colonization with S. aureus by suppressing production of RNA extraction and analysis of genes expression. the Arg-quorum-sensing signaling system [21]. In light of recent evidence implicating anti-infection and decoloni- Planktonic bacterial growth assay zation role of Bacillus lipopeptides against S. aureus, we To determine the antibacterial efect of B. subtilis CFS investigated the efect of B. subtilis cell-free supernatant on S. aureus, the growth of planktonic S. aureus was (B. subtilis CFS) on the growth of S. aureus in vitro and assessed using the method as described previously [23] in vivo. with some modifcations. Briefy, 100 µL of S. aureus sus- 8 Here we found that B. subtilis CFS exerted a potent pension (5 × ­10 CFU/mL) from a fresh overnight culture antimicrobial function against S. aureus and increased was inoculated into 5 mL TBS/PBS (1:1 v/v, control) or its susceptibility to antibiotics as well in vitro and in vivo TSB/B. subtilis CFS (1:1 v/v), and incubated with shaking as well. Furthermore, we demonstrated that B. subtilis at 200 rpm at 37 ℃. Te growth of S. aureus was deter- CFS changed the membrane components and increased mined by monitoring OD 600 of the cell culture at 2, 4, 6, membrane permeabilization of S. aureus, which may be 8, 10, 12 and 24 h after seeding. associated with increased susceptibility of S. aureus to antibiotics. Our data may suggest a potential application Bioflm formation and viability assay of bioflm S. aureus of B. subtilis CFS as an adjuvant to potentiate β-lactam To evaluate the efect of B. subtilis CFS on S. aureus 8 antibiotics against S. aureus osteomyelitis. bioflm formation, 100 µL of S. aureus (5 × ­10 CFU/ mL) was added to 900 µL of TSB/PBS (1:1 v/v), TSB/B. Materials and methods subtilis CFS (1:1 v/v), TSB/PBS (1:1 v/v) with 32 µg/mL Bacterial strains and culture penicillin, or TSB/PBS (1:1 v/v) with 0.75 µg/mL gen- Staphylococcus aureus strains were isolated from the tamicin in each well on a 24-well plate and incubated at osteomyelitis subjects from Department of Orthopedics, 37℃ for indicated time points without shaking. Next, Nanfang Hospital, Southern Medical University, using after the medium removed, the wells were washed three PHOENIX 100 (Becton Dickinson Microbiology Sys- times with sterile PBS. Finally, the plates were air-dried tem, USA). B. subtilis (CMCC-B-63,501) was obtained for 45 min and the adherent cells and matrix were stained from China General Microbiological Culture Collec- with 0.1 % crystal violet solution. To quantify the bioflm tion Center. Bacterial strains were cultured in TSB (Cat. production, crystal violet was extracted by incubation LA0110, Solarbio, Beijing, China) at 37 ℃ under shaking in solution (95 % ethanol and 0.1 % acetic acid) at room at 200 rpm. Overnight bacterial cultures were collected temperature for 15 min, and absorbance was measured at by a centrifuge, and pellets washed and resuspended in 600 nm in a microplate reader. phosphate-bufered saline (PBS) (Cat. C10010500BT, SYTO9 (Cat. S34854, Invitrogen, Termo Fisher Scien- GIBCO, Beijing, China). Te bacterial suspensions were tifc) and propidium iodide (PI) (Cat. P346, DOJINDO, Zhang et al. Microb Cell Fact (2021) 20:102 Page 3 of 15 Japan) staining was performed to evaluate the efect of B.
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