Shota Higashihira1,2,3,4, Stefanie Jan Simpson1,2, Christopher David Collier1,2, Roman Michael Natoli1,2, Mizuho Kittaka2,4, Edward Michael Greenfield1,2. 1. Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN, USA. 2. Indiana Center for Musculoskeletal Health, Indiana University School of Medicine, Indianapolis, IN, USA. 3. Department of Orthopaedic Surgery, Yokohama City University, Yokohama, Japan. 4. Department of Biomedical Sciences and Comprehensive Care, Indiana University School of Dentistry, Indianapolis, IN, USA.
Abstract
BACKGROUND: Biofilms protect bacteria from the host immune system and many antibiotics, making the treatment of orthopaedic infections difficult. Halicin, a recently discovered antibiotic, has potent activity against nonorthopaedic infections in mice and the planktonic, free-living forms of many bacterial species, including Staphylococcus aureus , a common cause of orthopaedic infections. Importantly, halicin did not induce resistance in vitro and was effective against drug-resistant bacteria and proliferating and quiescent bacteria. Quiescence is an important cause of antibiotic tolerance in biofilms. However, whether halicin acts on biofilms has not been tested. QUESTIONS/PURPOSES: (1) Does halicin reduce the viability of S. aureus in less mature and more mature biofilms as it does in planktonic cultures? (2) How do the relative effects of halicin on S. aureus biofilms and planktonic cultures compare with those of conventional antibiotics (tobramycin, cefazolin, vancomycin, or rifampicin) that are commonly used in clinical orthopaedic infections? METHODS: To measure minimal biofilm eradication concentrations (MBECs) with less mature 3-day and more mature 7-day biofilms, we used 96-well peg plates that provided high throughput and excellent reproducibility. After S. aureus -Xen36 biofilm formation, planktonic bacteria were removed from the cultures, and the biofilms were exposed to various concentrations of halicin, tobramycin, cefazolin, vancomycin, or rifampicin for 20 hours. Biofilm viability was determined by measuring resazurin reduction or by counting colony-forming units after sonication. To determine effects of halicin and the conventional antibiotics on biofilm viability, we defined MBEC 75 as the lowest concentration that decreased viability by 75% or more. To determine effects on bacterial viability in planktonic cultures, minimum inhibitory concentrations (MICs) were determined with the broth dilution method. Each result was measured in four to 10 independent experiments. RESULTS: We found no differences between halicin's effectiveness against planktonic S. aureus and 3-day biofilms (MIC and MBEC 75 for 3-day biofilms was 25 μM [interquartile range 25 to 25 and 25 to 25, respectively]; p > 0.99). Halicin was eightfold less effective against more mature 7-day biofilms (MBEC 75 = 200 μM [100 to 200]; p < 0.001). Similarly, tobramycin was equally effective against planktonic culture and 3-day biofilms (MIC and MBEC 75 for 3-day biofilms was 20 μM [20 to 20 and 10 to 20, respectively]; p > 0.99). Tobramycin's MBEC 75 against more mature 7-day biofilms was 320 μM (320 to 480), which is 16-fold greater than its planktonic MIC (p = 0.03). In contrast, the MBEC 75 for cefazolin, vancomycin, and rifampicin against more mature 7-day biofilms were more than 1000-fold (> 1000; p < 0.001), 500-fold (500 to 875; p < 0.001), and 3125-fold (3125 to 5469; p = 0.004) greater than their planktonic MICs, respectively, consistent with those antibiotics' relative inactivity against biofilms. CONCLUSION: Halicin was as effective against S. aureus in less mature 3-day biofilms as those in planktonic cultures, but eightfold higher concentrations were needed for more mature 7-day biofilms. Tobramycin, an antibiotic whose effectiveness depends on biofilm maturity, was also as effective against S. aureus in less mature 3-day biofilms as those in planktonic cultures, but 16-fold higher concentrations were needed for more mature 7-day biofilms. In contrast, cefazolin, vancomycin, and rifampicin were substantially less active against both less and more mature biofilms than against planktonic cultures. CLINICAL RELEVANCE: Halicin is a promising antibiotic that may be effective against S. aureus osteomyelitis and infections on orthopaedic implants. Future studies should assess the translational value of halicin by testing its effects in animal models of orthopaedic infections; on the biofilms of other bacterial species, including multidrug-resistant bacteria; and in combination therapy with conventional antibiotics.
BACKGROUND: Biofilms protect bacteria from the host immune system and many antibiotics, making the treatment of orthopaedic infections difficult. Halicin, a recently discovered antibiotic, has potent activity against nonorthopaedic infections in mice and the planktonic, free-living forms of many bacterial species, including Staphylococcus aureus , a common cause of orthopaedic infections. Importantly, halicin did not induce resistance in vitro and was effective against drug-resistant bacteria and proliferating and quiescent bacteria. Quiescence is an important cause of antibiotic tolerance in biofilms. However, whether halicin acts on biofilms has not been tested. QUESTIONS/PURPOSES: (1) Does halicin reduce the viability of S. aureus in less mature and more mature biofilms as it does in planktonic cultures? (2) How do the relative effects of halicin on S. aureus biofilms and planktonic cultures compare with those of conventional antibiotics (tobramycin, cefazolin, vancomycin, or rifampicin) that are commonly used in clinical orthopaedic infections? METHODS: To measure minimal biofilm eradication concentrations (MBECs) with less mature 3-day and more mature 7-day biofilms, we used 96-well peg plates that provided high throughput and excellent reproducibility. After S. aureus -Xen36 biofilm formation, planktonic bacteria were removed from the cultures, and the biofilms were exposed to various concentrations of halicin, tobramycin, cefazolin, vancomycin, or rifampicin for 20 hours. Biofilm viability was determined by measuring resazurin reduction or by counting colony-forming units after sonication. To determine effects of halicin and the conventional antibiotics on biofilm viability, we defined MBEC 75 as the lowest concentration that decreased viability by 75% or more. To determine effects on bacterial viability in planktonic cultures, minimum inhibitory concentrations (MICs) were determined with the broth dilution method. Each result was measured in four to 10 independent experiments. RESULTS: We found no differences between halicin's effectiveness against planktonic S. aureus and 3-day biofilms (MIC and MBEC 75 for 3-day biofilms was 25 μM [interquartile range 25 to 25 and 25 to 25, respectively]; p > 0.99). Halicin was eightfold less effective against more mature 7-day biofilms (MBEC 75 = 200 μM [100 to 200]; p < 0.001). Similarly, tobramycin was equally effective against planktonic culture and 3-day biofilms (MIC and MBEC 75 for 3-day biofilms was 20 μM [20 to 20 and 10 to 20, respectively]; p > 0.99). Tobramycin's MBEC 75 against more mature 7-day biofilms was 320 μM (320 to 480), which is 16-fold greater than its planktonic MIC (p = 0.03). In contrast, the MBEC 75 for cefazolin, vancomycin, and rifampicin against more mature 7-day biofilms were more than 1000-fold (> 1000; p < 0.001), 500-fold (500 to 875; p < 0.001), and 3125-fold (3125 to 5469; p = 0.004) greater than their planktonic MICs, respectively, consistent with those antibiotics' relative inactivity against biofilms. CONCLUSION: Halicin was as effective against S. aureus in less mature 3-day biofilms as those in planktonic cultures, but eightfold higher concentrations were needed for more mature 7-day biofilms. Tobramycin, an antibiotic whose effectiveness depends on biofilm maturity, was also as effective against S. aureus in less mature 3-day biofilms as those in planktonic cultures, but 16-fold higher concentrations were needed for more mature 7-day biofilms. In contrast, cefazolin, vancomycin, and rifampicin were substantially less active against both less and more mature biofilms than against planktonic cultures. CLINICAL RELEVANCE: Halicin is a promising antibiotic that may be effective against S. aureus osteomyelitis and infections on orthopaedic implants. Future studies should assess the translational value of halicin by testing its effects in animal models of orthopaedic infections; on the biofilms of other bacterial species, including multidrug-resistant bacteria; and in combination therapy with conventional antibiotics.
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