Real-Time Impedance-Based Monitoring of the Growth and Inhibition of Osteomyelitis Biofilm Pathogen Staphylococcus aureus Treated with Novel Bisphosphonate-Fluoroquinolone Antimicrobial Conjugates.

Osteomyelitis is a limb- and life-threatening orthopedic infection predominantly caused by Staphylococcus aureus biofilms. Bone infections are extremely challenging to treat clinically. Therefore, we have been designing, synthesizing, and testing novel antibiotic conjugates to target bone infections. This class of conjugates comprises bone-binding bisphosphonates as biochemical vectors for the delivery of antibiotic agents to bone minerals (hydroxyapatite). In the present study, we utilized a real-time impedance-based assay to study the growth of Staphylococcus aureus biofilms over time and to test the antimicrobial efficacy of our novel conjugates on the inhibition of biofilm growth in the presence and absence of hydroxyapatite. We tested early and newer generation quinolone antibiotics (ciprofloxacin, moxifloxacin, sitafloxacin, and nemonoxacin) and several bisphosphonate-conjugated versions of these antibiotics (bisphosphonate-carbamate-sitafloxacin (BCS), bisphosphonate-carbamate-nemonoxacin (BCN), etidronate-carbamate-ciprofloxacin (ECC), and etidronate-carbamate-moxifloxacin (ECX)) and found that they were able to inhibit Staphylococcus aureus biofilms in a dose-dependent manner. Among the conjugates, the greatest antimicrobial efficacy was observed for BCN with an MIC of 1.48 µg/mL. The conjugates demonstrated varying antimicrobial activity depending on the specific antibiotic used for conjugation, the type of bisphosphonate moiety, the chemical conjugation scheme, and the presence or absence of hydroxyapatite. The conjugates designed and tested in this study retained the bone-binding properties of the parent bisphosphonate moiety as confirmed using high-performance liquid chromatography. They also retained the antimicrobial activity of the parent antibiotic in the presence or absence of hydroxyapatite, albeit at lower levels due to the nature of their chemical modification. These findings will aid in the optimization and testing of this novel class of drugs for future applications to pharmacotherapy in osteomyelitis.

Bisphosphonates in dentistry: Historical perspectives, adverse effects, and novel applications.

Studies of the potential role of bisphosphonates in dentistry date back to physical chemical research in the 1960s, and the genesis of the discovery of bisphosphonate pharmacology in part can be linked to some of this work. Since that time, parallel research on the effects of bisphosphonates on bone metabolism continued, while efforts in the dental field included studies of bisphosphonate effects on dental calculus, caries, and alveolar bone loss. While some utility of this drug class in the dental field was identified, leading to their experimental use in various dentrifice formulations and in some dental applications clinically, adverse effects of bisphosphonates in the jaws have also received attention. Most recently, certain bisphosphonates, particularly those with strong bone targeting properties, but limited biochemical effects (low potency bisphosphonates), are being studied as a local remedy for the concerns of adverse effects associated with other more potent members of this drug class. Additionally, low potency bisphosphonate analogs are under study as vectors to target active drugs to the mineral surfaces of the jawbones. These latter efforts have been devised for the prevention and treatment of oral problems, such as infections associated with oral surgery and implants. Advances in the utility and mechanistic understanding of the bisphosphonate class may enable additional oral therapeutic options for the management of multiple aspects of dental health.

Bisphosphonates for delivering drugs to bone.

Advances in the design of potential bone-selective drugs for the treatment of various bone-related diseases are creating exciting new directions for multiple unmet medical needs. For bone-related cancers, off-target/non-bone toxicities with current drugs represent a significant barrier to the quality of life of affected patients. For bone infections and osteomyelitis, bacterial biofilms on infected bones limit the efficacy of antibiotics because it is hard to access the bacteria with current approaches. Promising new experimental approaches to therapy, based on bone-targeting of drugs, have been used in animal models of these conditions and demonstrate improved efficacy and safety. The success of these drug-design strategies bodes well for the development of therapies with improved efficacy for the treatment of diseases affecting the skeleton. LINKED ARTICLES: This article is part of a themed issue on The molecular pharmacology of bone and cancer-related bone diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.9/issuetoc.

Evaluation of 1,2-Benzothiazine 1,1-dioxide Derivatives In Vitro Activity towards Clinical-Relevant Microorganisms and Fibroblasts.

The global concern related with growing number of bacterial pathogens, resistant to numerous antibiotics, prone scientific environment to search for new antimicrobials. Antiseptics appear to be suitable candidates as adjunctive agents to antibiotics or alternative local treatment option aiming to prevent and treat infections. 1,2-benzothiazines are considered one the most promising of them. In this research twenty 1,2-benzothiazine 1,1-dioxide derivatives were scrutinized with regard to their biological activity. Three of them are new. For evaluation of compounds' activity against microbial pathogens, disk diffusion method and serial microdilution method was applied. To establish the cytotoxicity profile of tested 1,2-benzothiazines 1,1-dioxides derivatives, the cytotoxicity assay using fibroblasts L292 was performed. Antimicrobial activity of all tested compounds against Gram-positive Staphylococcus aureus and Enterococcus faecalis strains was higher than antimicrobial activity of DMSO solvent, which possesses antimicrobial activity itself. Gram-negative P. aeruginosa, E. coli and K. pneumoniae have shown susceptibility only to compounds 3e, 7i and 7l. None of tested compounds was effective against C. albicans. Compound 6g has demonstrated the strongest antimicrobial potency (MIC = 0.00975 mg/mL) among compounds of series 6. Compounds of series 7, namely 7d, 7f, 7g had the lowest minimum inhibitory concentration (MIC). Compound 7f displayed also the lowest cytotoxic effect against fibroblast cell line among series 7 compounds. All tested derivatives displayed lower MIC against Gram-positive bacteria than commercially applied antiseptic, povidone iodine, which MIC value range for tested Gram-positive bacteria was 1.56-6.25 mg/mL.

Bacterial cellulose as a support for yeast immobilization - Correlation between carrier properties and process efficiency.

The aim of the current study was to analyze physicochemical properties of bacterial cellulose (BC) produced by Komagataeibacter xylinus for various periods of time in stationary conditions with regard to its potential as a carrier for yeast (Saccharomyces cerevisiae and Yarrowia lipolytica) immobilization, and subsequently to correlate the relationship between these properties and the efficiency of the immobilization process. Physicochemical properties of BC, depending on the time of its biosynthesis, were as follows: surface area 7.21-11.04 m2/g, pore volume 3.11-3.96 cm3/g, pore diameter 0.011-0.109 nm, water holding capacity 32-64%, water relies capacity 10600-33400%, swelling ratio 132-389%, polymerization degree 2260-4780 and total crystallinity index 1.22-1.96 (3-30 days, respectively). The linear regression analysis showed that number of immobilized yeasts increased with values of surface area, pore size, pore diameter, swelling ratio, water release capacity and polymerization degree. The opposite trend was observed in case of water holding capacity and total crystallinity index. The analysis of physicochemical properties of BC performed in the current study have significant translational implications for understanding the relationships between BC-based carriers and the efficiency of yeast immobilization.

The influence of different composite mixtures (PLA/HA) manufactured with additive laser technology on the ability of S. aureus and P. aeruginosa to form biofilms.

PURPOSE: Staphylococcus aureus (Gram-positive coccus) and Pseudomonas aeruginosa (Gram-negative bacterium) are the leading etiologic agents of biofilm-related, life-threatening infections in patients after orthopaedic implantations. The aim of the present paper is to estimate the ability of these two bacterial strains to form a biofilm on bioresorbable composites manufactured from polylactide (PLA) and hydroxyapatite (HA) with the use of Selective Laser Sintering (SLS) method. METHODS: Microbiological tests were conducted on two variants of a solid specimen made with additive laser technology. Samples with different content of hydroxyapatite were made, with appropriate manufacturing parameters to ensure stability of both composite ingredients. The geometry of samples was obtained by tech- nical computed tomography. Microbiological tests determined the number of bacterial cells after incubation. RESULTS: The results indicate significantly decreased ability of S. aureus and P. aeruginosa to form biofilms on the surface of materials with higher content of hy- droxyapatite ceramics. CONCLUSIONS: The data may be useful for future applications of SLS technology in the production of bioresorbable PLA/HA medical implants.

Bacterial cellulose yield increased over 500% by supplementation of medium with vegetable oil.

Bacterial cellulose (BC), produced by Komagataeibacter xylinus, has numerous applications to medicine and industry. A major limitation of BC use is relatively low production rates and high culturing media costs. By supplementing culture media with 1% vegetable oil, we achieved BC yield exceeding 500% over the yield obtained in standard media. BC properties were similar to cellulose cultured in standard methods with regard to cytotoxicity but displayed significantly higher water swelling capacity and mechanical strength. As we demonstrated herein, this significantly increased BC yield is the result of microscopic and macroscopic physiochemical processes reflecting a complex interaction between K. xylinus biophysiology, chemical processes of BC synthesis, and physiochemical forces between BC membranes, oil and culturing vessel walls. Our findings have significant translational implications to biomedical and clinical settings and can be transformative for the cellulose biopolymer industry.

Modification of Bacterial Cellulose with Quaternary Ammonium Compounds Based on Fatty Acids and Amino Acids and the Effect on Antimicrobial Activity.

In the present work, bacterial cellulose (BC) membranes have been modified with bioactive compounds based on long chain dimer of C18 linoleic acid, referred to as the dilinoleic acid (DLA) and tyrosine (Tyr), a natural amino acid capable of forming noncovalent cation-π interactions with positively charged ethylene diamine (EDA). This new compound, [EDA][DLA-Tyr], has been synthesized by simple coupling reaction, and its chemical structure was characterized by 1H NMR and Fourier transform infrared spectroscopy. The antimicrobial activity of a new compound against S. aureus and S. epidermidis, two cocci associated with skin and wound infections, was assessed. The [EDA][DLA-Tyr] impregnated BC exhibited strong and long-term antimicrobial activity against both staphylococcal species. The results showed a 57-66% and 56-60% reduction in S. aureus and S. epidermidis viability, respectively, depending on [EDA][DLA-Tyr] concentration used. Importantly, [EDA][DLA-Tyr] molecules were released gradually from the BC pellicle, while a reference antibiotic, erythromycine (ER), did not show any antibacterial activity against S. aureus and S. epidermidis after 48 h of soaking in deionized water. Thus, a combination of [EDA][DLA-Tyr] and BC could be a promising new class of wound dressing displaying both biocompatibility and antimicrobial activity.

A.D.A.M. test (Antibiofilm Dressing's Activity Measurement) - Simple method for evaluating anti-biofilm activity of drug-saturated dressings against wound pathogens.

In the present article, we propose a simple Antibiofilm Dressing's Activity Measurement (A.D.A.M.) test that allows to check in vitro a dressing's suitability against biofilm-related wound infections. To perform the test, three agar discs are covered with biofilm formed by the tested pathogen after which they are assembled one over another in the form of an agar plug and placed in the well of a 24-well plate. The top disc is covered with the analyzed dressing and the entire set is incubated for 24h. During this time, the investigated antimicrobial substance is released from the dressing and penetrates to subsequent biofilm-covered agar discs. Biofilm reduction is measured using 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) spectrometric assay and the results are compared to untreated control samples (agar plug covered with biofilm and without the dressing/or with a passive dressing placed on the top disc). Furthermore, in order to standardize the differences in penetrability of the drugs released from active dressings the results can be expressed as a dimensionless value referred to as the Penetrability Index. In summary, A.D.A.M. test is simple, cheap, can be performed practically in every clinical laboratory and takes no more time than routine microbiological diagnostics. Apart from measuring the released drug's activity, the A.D.A.M. test allows to assess drug penetrability (across three agar discs), reflecting real wound conditions, where microbes are frequently hidden under the necrotic tissue or cloth. In conclusion, the A.D.A.M. test produces a high volume of data that, when analyzed, can provide a researcher with a valuable hint concerning the applicability of active dressings against specific biofilm pathogens in a particular setting.

Design, Synthesis, and Antimicrobial Evaluation of a Novel Bone-Targeting Bisphosphonate-Ciprofloxacin Conjugate for the Treatment of Osteomyelitis Biofilms.

Osteomyelitis is a major problem worldwide and is devastating due to the potential for limb-threatening sequelae and mortality. Osteomyelitis pathogens are bone-attached biofilms, making antibiotic delivery challenging. Here we describe a novel osteoadsorptive bisphosphonate-ciprofloxacin conjugate (BV600022), utilizing a "target and release" chemical strategy, which demonstrated a significantly enhanced therapeutic index versus ciprofloxacin for the treatment of osteomyelitis in vivo. In vitro antimicrobial susceptibility testing of the conjugate against common osteomyelitis pathogens revealed an effective bactericidal profile and sustained release of the parent antibiotic over time. Efficacy and safety were demonstrated in an animal model of periprosthetic osteomyelitis, where a single dose of 10 mg/kg (15.6 µmol/kg) conjugate reduced the bacterial load by 99% and demonstrated nearly an order of magnitude greater activity than the parent antibiotic ciprofloxacin (30 mg/kg, 90.6 µmol/kg) given in multiple doses. Conjugates incorporating a bisphosphonate and an antibiotic for bone-targeted delivery to treat osteomyelitis biofilm pathogens constitute a promising approach to providing high bone-antimicrobial potency while minimizing systemic exposure.

The chemical digestion of Ti6Al7Nb scaffolds produced by Selective Laser Melting reduces significantly ability of Pseudomonas aeruginosa to form biofilm.

In our previous work we reported the impact of hydrofluoric and nitric acid used for chemical polishing of Ti-6Al-7Nb scaffolds on decrease of the number of Staphylococcus aureus biofilm forming cells. Herein, we tested impact of the aforementioned substances on biofilm of Gram-negative microorganism, Pseudomonas aeruginosa, dangerous pathogen responsible for plethora of implant-related infections. The Ti-6Al-7Nb scaffolds were manufactured using Selective Laser Melting method. Scaffolds were subjected to chemical polishing using a mixture of nitric acid and fluoride or left intact (control group). Pseudomonal biofilm was allowed to form on scaffolds for 24 hours and was removed by mechanical vortex shaking. The number of pseudomonal cells was estimated by means of quantitative culture and Scanning Electron Microscopy. The presence of nitric acid and fluoride on scaffold surfaces was assessed by means of IR and rentgen spetorscopy. Quantitative data were analysed using the Mann-Whitney test (P ≤ 0.05). Our results indicate that application of chemical polishing correlates with significant drop of biofilm-forming pseudomonal cells on the manufactured Ti-6Al-7Nb scaffolds ( p = 0.0133, Mann-Whitney test) compared to the number of biofilm-forming cells on non-polished scaffolds. As X-ray photoelectron spectroscopy revealed the presence of fluoride and nitrogen on the surface of scaffold, we speculate that drop of biofilm forming cells may be caused by biofilm-supressing activity of these two elements.

Rapid and specific detection, molecular epidemiology, and experimental virulence of the O16 subgroup within Escherichia coli sequence type 131.

Escherichia coli sequence type 131 (ST131), a widely disseminated multidrug-resistant extraintestinal pathogen, typically exhibits serotype O25b:H4. However, certain ST131 isolates exhibit serotype O16:H5 and derive from a phylogenetic clade that is distinct from the classic O25b:H4 ST131 clade. Both clades are assigned to ST131 by the Achtman multilocus sequence typing (MLST) system and a screening PCR assay that targets ST131-specific sequence polymorphisms in the mdh and gyrB genes. However, they are classified as separate STs by the Pasteur Institute MLST system, and an ST131 PCR method that targets the O25b rfb region and an ST131-specific polymorphism in pabB detects only the O25b-associated clade. Here, we describe a novel PCR-based method that allows for rapid and specific detection of the O16-associated ST131 clade. The clade members uniformly contained allele 41 of fimH (type 1 fimbrial adhesin) and a narrow range of alleles of gyrA and parC (fluoroquinolone target genes). The virulence genotypes of the clade members resembled those of classic O25b:H4 ST131 isolates; representative isolates were variably lethal in a mouse subcutaneous sepsis model. Several pulsotypes spanned multiple sources (adults, children, pets, and human fecal samples) and locales. An analysis of recent clinical E. coli collections showed that the O16 ST131 clade is globally distributed, accounts for 1 to 5% of E. coli isolates overall, and, when compared with other ST131 isolates, it is associated with resistance to ampicillin, gentamicin, and trimethoprim-sulfamethoxazole and with susceptibility to fluoroquinolones and extended-spectrum cephalosporins. Attention to this O16-associated ST131 clade, which is facilitated by our novel PCR-based assay, is warranted in future epidemiological studies of ST131 and, conceivably, in clinical applications.