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Rafał Pokrowiecki,1,2 Urszula Szałaj,3,4 Damian Fudala,3 Tomasz Zaręba,5 Jacek Wojnarowicz,3 Witold Łojkowski,3 Stefan Tyski,5 Krzysztof Dowgierd,6 Agnieszka Mielczarek7 1Department of Cranio- Maxillofacial Surgery, Oral Surgery and Implantology, Medical University of Warsaw, Warsaw, Poland; 2Head and Neck Surgery Department—Maxillofacial Surgery Department, Craniofacial Center, Regional Specialized Children’s Hospital, Olsztyn, 10-561, Poland; 3Institute of High Pressure Physics, Polish Academy of Sciences, Warsaw, Poland; 4Faculty of Materials Engineering, Warsaw University of Technology, Warsaw, Poland; 5Department of Antibiotics and Microbiology, National Medicines Institute, Warsaw, Poland; 6Head and Neck Surgery Clinic for Children and Young Adults, Department of Clinical Pediatrics, University of Warmia and Mazury, Olsztyn, 10-719, Poland; 7Department of Conservative Dentistry, Medical University of Warsaw, Warsaw, PolandCorrespondence: Agnieszka Mielczarek, Department of Conservative Dentistry, Medical University of Warsaw, Warsaw, Poland, Tel +48 691226414 ; +48 22 116 64 46, Email agnieszka.mielczarek.wum@gmail.comBackground: Periimplantitis is continuously one of major threats for the uneventful functioning of dental implants. Current approaches of drug delivery systems are being more commonly implemented into oral- and maxillofacial biomaterials in order to decrease the risk of implant failure due to bacterial infection. Silver nanoparticles and their compounds have been proven in eradicating oral bacteria responsible for peri-implant infections. Nevertheless, their evaluation as coating for implant abutments has not been extensively evaluated so far. This article describes a novel coating consisting of zinc oxide (ZnO) and silver (Ag) nanoparticles (NPs). This coating was used to modify healing abutments that could be used as drug delivery systems in oral implantology.Materials and Method: Nanoparticles with a ZnO + 0.1% Ag composition were produced by microwave solvothermal synthesis and then incorporated into the surface of titanium healing abutments by high-power ultrasonic deposition. Surface morphology, roughness, wettability were evaluated. Ability of biofilm formation inhibition was tested against S. mutans, S. oralis, S. aureus and E. coli.Results: ZnO+0.1%Ag NPs were sufficiently deposed on the surface of the abutments creating nanostructured coating which increased surface roughness and decreased wettability. Modified abutments significantly decreased bacterial biofilm formation. Bacteria present in SEM studies were unlikely to settle and replicate on the experimental abutments as their cells were rounded, insufficiently spread on the surface and covered with released NPs.Conclusion: Experimental nanostructured abutments were easily manufactured by high-power ultrasonic deposition and provided significant antibacterial properties. Such biomaterials could be used as temporary drug delivery abutments for prevention and treatment of intra- and extraoral peri-implant infections in the area of the head and neck.Keywords: dental implants, periimplantitis, nanotechnology, nanoparticles, NPs, drug delivery, zinc oxide, ZnO, silver, Ag

Rafal Pokrowiecki,1–3 Jacek Wojnarowicz,4 Tomasz Zareba,2 Iwona Koltsov,4 Witold Lojkowski,4 Stefan Tyski,2,5 Agnieszka Mielczarek,6 Pawel Zawadzki1 1Department of Cranio-Maxillofacial Surgery, Oral Surgery and Implantology, Medical University of Warsaw, Warsaw, Poland; 2Department of Antibiotics and Microbiology, National Medicines Institute, Warsaw, Poland; 3Private Practice, Warsaw, Poland; 4Institute of High Pressure Physics, Polish Academy of Sciences, Warsaw, Poland; 5Department of Pharmaceutical Microbiology, Medical University of Warsaw, Warsaw, Poland; 6Department of Conservative Dentistry, Medical University of Warsaw, Warsaw, PolandCorrespondence: Agnieszka MielczarekDepartment of Conservative Dentistry, Medical University of Warsaw, Binieckiego 6, Warsaw 02-097, PolandTel +48 22 116 6446Fax +48 572 0154Email sekretariat.zachowawcza@wum.edu.plAim: The aims of this study were to investigate new nano-formulations based on ZnO and Ag nanoparticle (NP) compounds when used against clinical strains of oral gram-positive and gram-negative bacteria, and to examine the stability and behaviour of nano-formulation mixtures in saliva based on different compositions of Ag NPs, ZnO NPs and ZnO+x·Ag NPs. Methods: ZnO NPs with and without nanosilver were obtained by microwave solvothermal synthesis. Then, antibacterial activity was evaluated against bacteria isolated from human saliva. Behavior and nanoparticle solutions were evaluated in human saliva and control (artificial saliva and deionized water). Results were statistically compared. Results: The NP mixtures had an average size of 30±3 nm, while the commercial Ag NPs had an average size of 55±5 nm. The suspensions displayed differing antibacterial activities and kinetics of destabilisation processes, depending on NPs composition and fluid types.Conclusion: The present study showed that all NPs suspensions displayed significant destabilisation and high destabilisation over the 24 h of the analyses. The agglomeration processes of NPs in human saliva can be reversible.Keywords: nanomaterials, controlled drug release, nanoparticles, metal nanoparticles, bacteria

Rafal Pokrowiecki,1 Agnieszka Mielczarek,2 Tomasz Zareba,3 Stefan Tyski3,4 1Department of Head and Neck Surgery-Maxillofacial Surgery, Otolaryngology and Ophthalmology, Prof Stanislaw Popowski Voivoid Children Hospital, Olsztyn, 2Department of Conservative Dentistry, Medical University of Warsaw, 3Department of Antibiotics and Microbiology, National Medicines Institute, 4Department of Pharmaceutical Microbiology, Medical University of Warsaw, Warsaw, Poland Abstract: Peri-implant infective diseases (PIIDs) in oral implantology are commonly known as peri-implant mucositis (PIM) and periimplantitis (PI). While PIM is restricted to the peri-implant mucosa and is reversible, PI also affects implant-supporting bone and, therefore, is very difficult to eradicate. PIIDs in clinical outcome may resemble gingivitis and periodontitis, as they share similar risk factors. However, recent study in the field of proteomics and other molecular studies indicate that PIIDs exhibit significant differences when compared to periodontal diseases. This review aims to elucidate the current knowledge of PIIDs, their etiopathology and diversified microbiology as well as the role of molecular studies, which may be a key to personalized diagnostic and treatment protocols of peri-implant infections in the near future. Keywords: dental plaque, infection, titanium, microbiome, periimplantitis

Rafał Pokrowiecki,1,2 Tomasz Zaręba,3 Barbara Szaraniec,4 Krzysztof Pałka,5 Agnieszka Mielczarek,6 Elżbieta Menaszek,7 Stefan Tyski3,8 1Center for Cranio-Maxillo-Facial Surgery, Voivodeship Children’s Hospital, Olsztyn, 2Department of Oral Surgery, Jagiellonian Medical University, Kraków, 3Department of Antibiotics and Microbiology, National Medicines Institute, Warsaw, 4Faculty of Material Science and Ceramics, AGH University of Science and Technology, Kraków, 5Department of Materials Engineering, Lublin University of Technology, Lublin, 6Department of Conservative Dentistry, Medical University of Warsaw, Warsaw, 7Department of Cytobiology, Collegium Medicum, Jagiellonian University, Kraków, 8Department of Pharmaceutical Microbiology, Medical University of Warsaw, Warsaw, Poland Abstract: The addition of an antibacterial agent to dental implants may provide the opportunity to decrease the percentage of implant failures due to peri-implantitis. For this purpose, in this study, the potential efficacy of nanosilver-doped titanium biomaterials was determined. Titanium disks were incorporated with silver nanoparticles over different time periods by Tollens reaction, which is considered to be an eco-friendly, cheap, and easy-to-perform method. The surface roughness, wettability, and silver release profile of each disc were measured. In addition, the antibacterial activity was also evaluated by using disk diffusion tests for bacteria frequently isolated from the peri-implant biofilm: Streptococcus mutans, Streptococcus mitis, Streptococcus oralis, Streptococcus sanguis, Porphyromonas gingivalis, Staphylococcus aureus, and Escherichia coli. Cytotoxicity was evaluated in vitro in a natural human osteoblasts cell culture. The addition of nanosilver significantly increased the surface roughness and decreased the wettability in a dose-dependent manner. These surfaces were significantly toxic to all the tested bacteria following a 48-hour exposure, regardless of silver doping duration. A concentration of 0.05 ppm was sufficient to inhibit Gram-positive and Gram-negative species, with the latter being significantly more susceptible to silver ions. However, after the exposure of human osteoblasts to 0.1 ppm of silver ions, a significant decrease in cell viability was observed by using ToxiLight™ BioAssay Kit after 72 hours. Data from the present study indicated that the incorporation of nanosilver may influence the surface properties that are important in the implant healing process. The presence of nanosilver on the titanium provides an antibacterial activity related to the bacteria involved in peri-implantitis. Finally, the potential toxicological considerations of nanosilver should further be investigated, as both the antibacterial and cytotoxic properties may be observed at similar concentration ranges. Keywords: biomaterials, dental plaque, peri-implantitis, peri implant mucositis, silver, nanotechnology, nanomedicine

Disinfectants and antiseptics lead in reducing the number of microorganisms, including pathogenic ones, thus limiting the number of infections. In the veterinary field, disinfection prevents the transfer of pathogenic microorganisms from animals to humans and vice versa, as well as among animals. Several assays of disinfectant antimicrobial activity testing, often not standardized, without appropriate controls, and not validated, have been used and published. To unify these methods, nine European Standards (ENs) for the veterinary area have been prepared. These tests make it possible to examine whether a given disinfectant has bactericidal, fungicidal, or virucidal activity by the standard. This publication discusses ENs regarding the assessment of the above-mentioned antimicrobial activity of disinfectants used in veterinary medicine. Recent research on this topic has also been cited. According to ENs, tests are carried out using the suspension method or carriers in clean and dirty conditions. The decontamination of high-risk animal and zoonotic pathogens is also discussed. Selected publications on cattle, pig, poultry, and aquaculture farm disinfection are presented. Only valid methods of the described studies with appropriate statistical analysis can prove adequate antimicrobial activity. So far, the role of international standards in investigating the antimicrobial activity of disinfectants and antiseptics to reduce infections has been underestimated. This publication highlights gaps and irregularities in conducted research and aims to inform about existing EN standards dedicated to testing the biocidal activity of disinfectants and antiseptics intended for use in the veterinary area.

Transplantology is a branch of medicine that is developing rapidly. Transplanted whole organs or segments of organs may be recovered from either living or deceased donors. New methods of preserving transplanted solid organs, including the kidney, liver, heart, lung, and pancreas, as well as tissues, such as the cornea and skin, are being developed. Preservation fluid, which is used to perfuse and store the donated organ or tissue, should reduce biological deterioration of organs and tissue, attenuate ischemia/reperfusion-induced cell/tissue injury, and protect against damage. Lowering the storage temperature of organs significantly reduces the risk of damage. Efforts are also made to shorten the time between collecting the organ or tissue from the donor and transplanting it in the recipient. However, during transplantation, the recipient may become infected, primarily with bacteria and fungi. Infections of organ recipients occur most often due to unhygienic organ collection, improper handling and transport, and inappropriate preservation conditions, especially contamination of preservation fluid. The literature on contamination of organ preservation fluid and infections in graft recipients is very diverse, both in terms of the isolated bacterial and fungal species and the number of incidents. A large percentage of contaminating microorganisms belong to the generally non-pathogenic skin microbiota, but there are also cases of multidrug-resistant bacteria. Besides, the transplanted organs themselves may pose a danger. They may contain latent microorganisms, mainly viruses and parasites, that could be activated in a patient who has been subjected to immunosuppression to reduce the risk of organ rejection.

Non-steroidal anti-inflammatory drugs (NSAIDs) commonly used in clinical practice may cause gastrointestinal injuries and influence the gut microbiota. This study investigated the effects of various NSAIDs and some analgesics on the viability of Lactobacillaceae strains (including probiotic strains) in vitro. It was found that diclofenac, ibuprofen, ketoprofen, dexketoprofen, flurbiprofen, and acetylsalicylic acid inhibited the growth of lactobacilli at a concentration of 0.05−3.2 mg/ml. These MICs of NSAIDs are well above therapeutic plasma concentrations achieved in humans, indicating that the tested drugs should not inhibit the growth of lactobacilli in the human digestive tract.

Lactic acid bacteria are used in various types of probiotic products. Due to the constantly growing probiotics market, new strains with pro-health properties are sought. The present study compared 39 strains of Lactobacillus, Lacticaseibacillus, and Lactiplantibacillus, isolated from probiotic products and healthy people. The current research aimed to search for new, potentially probiotic strains. For this purpose the relationship between Lactobacillaceae strains was carried out; moreover, the basic properties of probiotic microorganisms, such as survival at low pH and bile salt environment, antibiotic susceptibility, aggregation and antagonism were estimated. The properties of these isolates were also compared with the properties of probiotic strains from the ATCC collection. In comparing the genetic relationship (PFGE method) between the tested isolates, it was observed that some of them show a high degree of similarity. All tested strains tolerated an environment with a pH value of 3.0, and the addition of 0.3% bile salt; showed auto-aggregation properties and displayed antagonism against pathogenic microorganisms. In the present study, the bacteria were susceptible to tetracycline, chloramphenicol and ampicillin; the resistance to vancomycin

Cross-infection involves the transmission of microorganisms through secretions, bodily fluids and excreta, as well as undisinfected surfaces and medical equipment. In the dental office, diseases are transmitted via various routes, e.g. from patient to dentist or other member of dental team, from doctor or dental team member to patient, from patient to another patient, from dental office to community and from community to patient. The study was conducted to evaluate the effectiveness of infection control in dental practices based on the qualitative and quantitative assessment of microbiological contaminants detected on the surface of filling material packaging used in dental offices. The material for research were 9 packages containing dental materials during their use in 3 dental settings. The packages were placed in sterile flasks and rinsed to wash microorganisms from the surfaces. The washes were filtered through membrane filters and cultured under proper aerobic and anaerobic conditions, and at elevated CO2 concentration. Microbial growth on TIO and TSB media was observed. The contamination of most samples remained low as indicated by the growth from one to a maximum of five colonies on TSA. The contamination remained at the level of 10-50 CFU/package, i.e.

Titres of IgG antibodies to Staphylococcus aureus lipase were analysed in 448 sera from patients suspected of having Staphylococcus aureus infections and the results compared to those for the routinely used staphylococcal antigens teichoic acid and alpha-toxin. The results indicated that determination of serum antibodies to lipase is a sensitive assay for serological diagnosis of staphylococcal infections and increased sensitivity may be achieved by selection of optimal antigen combinations. [ABSTRACT FROM AUTHOR]

Despite the urgent need for new antibiotics, very few innovative antibiotics have recently entered clinics or clinical trials. To provide a constant supply of new drug candidates optimized in terms of their potential to select for resistance in natural settings, in vitro resistance-predicting studies need to be improved and scaled up. In this review, the following in vitro parameters are presented: frequency of spontaneous mutant selection (FSMS), mutant prevention concentration (MPC), dominant mutant prevention concentration (MPC-D), inferior-mutant prevention concentration (MPC-F), and minimal selective concentration (MSC). The utility of various adaptive laboratory evolution (ALE) approaches (serial transfer, continuous culture, and evolution in spatiotemporal microenvironments) for comparing hits in terms of the level and time required for multistep resistance to emerge is discussed. We also consider how the hit-to-lead stage can benefit from high-throughput ALE setups based on robotic workstations, do-it-yourself (DIY) continuous cultivation systems, microbial evolution and growth arena (MEGA) plates, soft agar gradient evolution (SAGE) plates, microfluidic chips, or microdroplet technology. Finally, approaches for evaluating the fitness of in vitro-generated resistant mutants are presented. This review aims to draw attention to newly emerged ideas on how to improve the in vitro forecasting of the potential of compounds to select for resistance in natural settings.

The efflux pumps, beside the class D carbapenem-hydrolysing enzymes (CHLDs), are being increasingly investigated as a mechanism of carbapenem resistance in Acinetobacter baumannii . This study investigates the contribution of efflux mechanism to carbapenem resistance in 61 acquired bla CHDL -genes-carrying A. baumannii clinical strains isolated in Warsaw, Poland. Studies were conducted using phenotypic (susceptibility testing to carbapenems ± efflux pump inhibitors (EPIs)) and molecular (determining expression levels of efflux operon with regulatory-gene and whole genome sequencing (WGS)) methods. EPIs reduced carbapenem resistance of 14/61 isolates. Upregulation (5-67-fold) of adeB was observed together with mutations in the sequences of AdeRS local and of BaeS global regulators in all 15 selected isolates. Long-read WGS of isolate no. AB96 revealed the presence of AbaR25 resistance island and its two disrupted elements: the first contained a duplicate IS Aba1-bla OXA-23 , and the second was located between adeR and adeA in the efflux operon. This insert was flanked by two copies of IS Aba1 , and one of them provides a strong promoter for adeABC , elevating the adeB expression levels. Our study for the first time reports the involvement of the insertion of the ΔAbaR25-type resistance island fragment with IS Aba1 element upstream the efflux operon in the carbapenem resistance of A. baumannii .

The analysis of antimicrobial activity is usually MIC- and minimal bactericidal concentration (MBC)-focused, though also crucial are resistance-related parameters, e.g., the frequency of spontaneous mutant selection (FSMS), the mutant prevention concentration (MPC), and the mutant selection window (MSW). In vitro -determined MPCs, however, are sometimes variable, poorly repeatable, and not always reproducible in vivo . We propose a new approach to the in vitro determination of MSWs, along with novel parameters: MPC-D, MSW-D (for dominant mutants, i.e., selected with a high frequency, without a fitness loss), and MPC-F, MSW-F (for inferior mutants, i.e., with an impaired fitness). We also propose a new method for preparing the high-density inoculum (>10 11 CFU/mL). In this study, the MPC and MPC-D (limited by FSMS of <10 -10 ) of ciprofloxacin, linezolid, and novel benzosiloxaborole (No37) were determined for Staphylococcus aureus ATCC 29213 using the standard agar method, while the MPC-D and MPC-F were determined by the novel broth method. Regardless of the method, MSWs 10 10 of linezolid and No37 were the same. However, MSWs 10 10 of ciprofloxacin in the broth method was narrower than in the agar method. In the broth method, the 24-h incubation of ~10 10 CFU in a drug-containing broth differentiates the mutants that can dominate the cell population from those that can only be selected under exposure. We consider MPC-Ds in the agar method to be less variable and more repeatable than MPCs. Meanwhile, the broth method may decrease discrepancies between in vitro and in vivo MSWs. The proposed approaches may help establish MPC-D-related resistance-restricting therapies., Competing Interests: The authors declare no conflict of interest.