Influence of microbiome species in hard-to-heal wounds on disease severity and treatment duration

ABSTRACT BACKGROUND: Infections, mostly those associated with colonization of wound by different pathogenic microorganisms, are one of the most serious health complications during a medical treatment. Therefore, this study is focused on the isolation, characterization, and identification of microorganisms prevalent in superficial wounds of patients (n = 50) presenting with bacterial infection. METHODS: After successful cultivation, bacteria were processed and analyzed. Initially the identification of the strains was performed through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry based on comparison of protein profiles (2-30 kDa) with database. Subsequently, bacterial strains from infected wounds were identified by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sequencing of 16S rRNA gene 108. RESULTS: The most prevalent species was Staphylococcus aureus (70%), and out of those 11% turned out to be methicillin-resistant (mecA positive). Identified strains were compared with patients' diagnoses using the method of artificial neuronal network to assess the association between severity of infection and wound microbiome species composition. Artificial neuronal network was subsequently used to predict patients' prognosis (n = 9) with 85% success. CONCLUSIONS: In all of 50 patients tested bacterial infections were identified. Based on the proposed artificial neuronal network we were able to predict the severity of the infection and length of the treatment.

Influence of microbiome species in hard-to-heal wounds on disease severity and treatment duration.

BACKGROUND: Infections, mostly those associated with colonization of wound by different pathogenic microorganisms, are one of the most serious health complications during a medical treatment. Therefore, this study is focused on the isolation, characterization, and identification of microorganisms prevalent in superficial wounds of patients (n=50) presenting with bacterial infection. METHODS: After successful cultivation, bacteria were processed and analyzed. Initially the identification of the strains was performed through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry based on comparison of protein profiles (2-30kDa) with database. Subsequently, bacterial strains from infected wounds were identified by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sequencing of 16S rRNA gene 108. RESULTS: The most prevalent species was Staphylococcus aureus (70%), and out of those 11% turned out to be methicillin-resistant (mecA positive). Identified strains were compared with patients' diagnoses using the method of artificial neuronal network to assess the association between severity of infection and wound microbiome species composition. Artificial neuronal network was subsequently used to predict patients' prognosis (n=9) with 85% success. CONCLUSIONS: In all of 50 patients tested bacterial infections were identified. Based on the proposed artificial neuronal network we were able to predict the severity of the infection and length of the treatment.

Staphylococcus aureus and MRSA Growth and Biofilm Formation after Treatment with Antibiotics and SeNPs.

Methicillin-resistant Staphylococcus aureus (MRSA) is a dangerous pathogen resistant to ß-lactam antibiotics. Due to its resistance, it is difficult to manage the infections caused by this strain. We examined this issue in terms of observation of the growth properties and ability to form biofilms in sensitive S. aureus and MRSA after the application of antibiotics (ATBs)-ampicillin, oxacillin and penicillin-and complexes of selenium nanoparticles (SeNPs) with these ATBs. The results suggest the strong inhibition effect of SeNPs in complexes with conventional ATBs. Using the impedance method, a higher disruption of biofilms was observed after the application of ATB complexes with SeNPs compared to the group exposed to ATBs without SeNPs. The biofilm formation was intensely inhibited (up to 99%±7% for S. aureus and up to 94%±4% for MRSA) after application of SeNPs in comparison with bacteria without antibacterial compounds whereas ATBs without SeNPs inhibited S. aureus up to 79%±5% and MRSA up to 16%±2% only. The obtained results provide a basis for the use of SeNPs as a tool for the treatment of bacterial infections, which can be complicated because of increasing resistance of bacteria to conventional ATB drugs.

Biological Activity and Molecular Structures of Bis(benzimidazole) and Trithiocyanurate Complexes.

1-(1H-Benzimidazol-2-yl)-N-(1H-benzimidazol-2-ylmethyl)methanamine (abb) and 2-(1H-benzimidazol-2-ylmethylsulfanylmethyl)-1H-benzimidazole (tbb) have been prepared and characterized by elemental analysis. These bis(benzimidazoles) have been further used in combination with trithiocyanuric acid for the preparation of complexes. The crystal and molecular structures of two of them have been solved. Each nickel atom in the structure of trinuclear complex [Ni3(abb)3(H2O)3(µ-ttc)](ClO4)3·3H2O·EtOH (1), where ttcH3 = trithiocyanuric acid, is coordinated with three N atoms of abb, the N,S donor set of ttc anion and an oxygen of a water molecule. The crystal of [(tbbH2)(ttcH2)2(ttcH3)(H2O)] (2) is composed of a protonated bis(benzimidazole), two ttcH2 anions, ttcH3 and water. The structure is stabilized by a network of hydrogen bonds. These compounds were primarily synthesized for their potential antimicrobial activity and hence their possible use in the treatment of infections caused by bacteria or yeasts (fungi). The antimicrobial and antifungal activity of the prepared compounds have been evaluated on a wide spectrum of bacterial and yeast strains and clinical specimens isolated from patients with infectious wounds and the best antimicrobial properties were observed in strains after the use of ligand abb and complex 1, when at least 80% growth inhibition was achieved.

Study of linkage between glutathione pathway and the antibiotic resistance of Escherichia coli from patients' swabs.

In this work, we focused on the differences between bacterial cultures of E. coli obtained from swabs of infectious wounds of patients compared to laboratory E. coli. In addition, blocking of the protein responsible for the synthesis of glutathione (γ-glutamylcysteine synthase-GCL) using 10 mM buthionine sulfoximine was investigated. Each E. coli showed significant differences in resistance to antibiotics. According to the determined resistance, E. coli were divided into experimental groups based on a statistical evaluation of their properties as more resistant and more sensitive. These groups were also used for finding the differences in a dependence of the glutathione pathway on resistance to antibiotics. More sensitive E. coli showed the same kinetics of glutathione synthesis while blocking GCL (Km 0.1 µM), as compared to non-blocking. In addition, the most frequent mutations in genes of glutathione synthetase, glutathione peroxidase and glutathione reductase were observed in this group compared to laboratory E.coli. The group of "more resistant" E. coli exhibited differences in Km between 0.3 and 0.8 µM. The number of mutations compared to the laboratory E. coli was substantially lower compared to the other group.

3D-printed chip for detection of methicillin-resistant Staphylococcus aureus labeled with gold nanoparticles.

Methicillin-resistant Staphylococcus aureus (MRSA) is a dangerous pathogen occurring not only in hospitals but also in foodstuff. Currently, discussions on the issue of the increasing resistance, and timely and rapid diagnostic of resistance strains have become more frequent and sought. Therefore, the aim of this study was to design an effective platform for DNA isolation from different species of microorganisms as well as the amplification of mecA gene that encodes the resistance to ß-lactam antibiotic formation and is contained in MRSA. For this purpose, we fabricated 3D-printed chip that was suitable for bacterial cultivation, DNA isolation, PCR, and detection of amplified gene using gold nanoparticle (AuNP) probes as an indicator of MRSA. Confirmation of the MRSA presence in the samples was based on a specific interaction between mecA gene with the AuNP probes and a colorimetric detection, which utilized the noncross-linking aggregation phenomenon of DNA-functionalized AuNPs. To test the whole system, we analyzed several real refractive indexes, in which two of them were positively scanned to find the presence of mecA gene. The aggregation of AuNP probes were reflected by 75% decrease of absorbance (λ = 530 nm) and change in AuNPs size from 3 ± 0.05 to 4 ± 0.05 nm (n = 5). We provide the one-step identification of mecA gene using the unique platform that employs the rapid, low-cost, and easy-to-use colorimetric method for MRSA detection in various samples.

The effect of metal ions on Staphylococcus aureus revealed by biochemical and mass spectrometric analyses.

In this study, we focused on the effect of heavy metal ions in resistant strains of gram-positive bacteria Staphylococcus aureus using biochemical methods and mass spectrometry. Five nitrate solutions of heavy metals (Ag(+), Cu(2+), Cd(2+), Zn(2+) and Pb(2+)) were used to create S. aureus resistant strains. Biochemical changes of resistant strains in comparison with the non-resistant control strain of S. aureus were observed by microbiological (measuring - growth curves and inhibition zones) and spectrophotometric methods (antioxidant activity and alaninaminotransferase, aspartateaminotransferase, alkaline phosphatase, γ-glutamyltransferase activities). Mass spectrometry was employed for the qualitative analysis of the samples (changes in S. aureus protein composition) and for the identification of the strains database MALDI Biotyper was employed. Alterations, in terms of biochemical properties and protein composition, were observed in resistant strains compared to non-resistant control strain. Our results describe the possible option for the analysis of S. aureus resistant strains and may thus serve as a support for monitoring of changes in genetic information caused by the forming of resistance to heavy metals.

Effect of ampicillin, streptomycin, penicillin and tetracycline on metal resistant and non-resistant Staphylococcus aureus.

There is an arising and concerning issue in the field of bacterial resistance, which is confirmed by the number of deaths associated with drug-resistant bacterial infections. The aim of this study was to compare the effects of antibiotics on Staphylococcus aureus non-resistant strain and strains resistant to cadmium or lead ions. Metal resistant strains were created by the gradual addition of 2 mM solution of metal ions (cadmium or lead) to the S. aureus culture. An increasing antimicrobial effect of ampicillin, streptomycin, penicillin and tetracycline (0, 10, 25, 50, 75, 150, 225 and 300 µM) on the resistant strains was observed using a method of growth curves. A significant growth inhibition (compared to control) of cadmium resistant cells was observed in the presence of all the four different antibiotics. On the other hand, the addition of streptomycin and ampicillin did not inhibit the growth of lead resistant strain. Other antibiotics were still toxic to the bacterial cells. Significant differences in the morphology of cell walls were indicated by changes in the cell shape. Our data show that the presence of metal ions in the urban environment may contribute to the development of bacterial strain resistance to other substances including antibiotics, which would have an impact on public health.

Remote-controlled robotic platform ORPHEUS as a new tool for detection of bacteria in the environment.

Remote-controlled robotic systems are being used for analysis of various types of analytes in hostile environment including those called extraterrestrial. The aim of our study was to develop a remote-controlled robotic platform (ORPHEUS-HOPE) for bacterial detection. For the platform ORPHEUS-HOPE a 3D printed flow chip was designed and created with a culture chamber with volume 600 µL. The flow rate was optimized to 500 µL/min. The chip was tested primarily for detection of 1-naphthol by differential pulse voltammetry with detection limit (S/N = 3) as 20 nM. Further, the way how to capture bacteria was optimized. To capture bacterial cells (Staphylococcus aureus), maghemite nanoparticles (1 mg/mL) were prepared and modified with collagen, glucose, graphene, gold, hyaluronic acid, and graphene with gold or graphene with glucose (20 mg/mL). The most up to 50% of the bacteria were captured by graphene nanoparticles modified with glucose. The detection limit of the whole assay, which included capturing of bacteria and their detection under remote control operation, was estimated as 30 bacteria per µL.

Fullerene as a transporter for doxorubicin investigated by analytical methods and in vivo imaging.

Carbon nanomaterials, including fullerenes, exhibit not only unique structure and electronic properties but also a significant potential to serve as radical scavengers and/or anti-oxidants. Their conjugation with anticancer drugs such as doxorubicin (DOX) may help to balance severe negative side effects of these cytostatics and also improve the delivery of the drug taking advantage of the enhanced cellular uptake, selectivity to cancer cells, and pH regulated release. In this study, the fullerene (C60) surface was oxidized by concentrated nitric acid, which enabled simple DOX-fullerene conjugation based on π-π stacking and hydrophilic interactions with carboxylic groups. The strength of this noncovalent binding is pH dependent. At a low pH, the amino group of DOX is protonated, however at a higher pH, the amino group is deprotonated, resulting in stronger hydrophobic interactions with the fullerene walls. CE and HPLC were employed for characterization of resulting complexes. The cell toxicity of the conjugates was evaluated using Staphylococcus aureus and finally they were administered into the chicken embryo to assess the applicability for in vivo imaging.

Comparison of the effects of silver phosphate and selenium nanoparticles on Staphylococcus aureus growth reveals potential for selenium particles to prevent infection.

Interactions of silver phosphate nanoparticles (SPNPs) and selenium nanoparticles (SeNPs) with Staphylococcus aureus cultures have been studied at the cellular, molecular and protein level. Significant antibacterial effects of both SPNPs and SeNPs on S. aureus were observed. At a concentration of 300 µM, SPNPs caused 37.5% inhibition of bacterial growth and SeNPs totally inhibited bacterial growth. As these effects might have been performed due to the interactions of nanoparticles with DNA and proteins, the interaction of SPNPs or SeNPs with the amplified zntR gene was studied. The presence of nanoparticles decreased the melting temperatures of the nanoparticle complexes with the zntR gene by 23% for SeNPs and by 12% for SPNPs in comparison with the control value. The concentration of bacterial metallothionein was 87% lower in bacteria after application of SPNPs (6.3 µg mg(-1) protein) but was increased by 29% after addition of SeNPs (63 µg mg(-1) protein) compared with the S. aureus control (49 µg mg(-1) protein). Significant antimicrobial effects of the nanoparticles on bacterial growth and DNA integrity provide a promising approach to reducing the risk of bacterial infections that cannot be controlled by the usual antibiotic treatments.

Lead ions encapsulated in liposomes and their effect on Staphylococcus aureus.

The aim of the study was the preparation of a liposome complex with encapsulated lead ions, which were electrochemically detected. In particular, experiments were focused on the potential of using an electrochemical method for the determination of free and liposome-encapsulated lead and determination of the encapsulation efficiency preventing the lead toxicity. Primarily, encapsulation of lead ions in liposomes and confirmation of successful encapsulation by electrochemical methods was done. Further, the reduction effect of the liposome matrix on the detected electrochemical signal was monitored. Besides encapsulation itself, comparison of toxicity of free lead ions and lead ions encapsulated in liposome was tested. The calculated IC50 values for evaluating the lead cytotoxicity showed significant differences between the lead enclosed in liposomes (28 µM) and free lead ions (237 µM). From the cytotoxicity studies on the bacterial strain of S. aureus it was observed that the free lead ions are less toxic in comparison with lead encapsulated in liposomes. Liposomes appear to be a suitable carrier of various substances through the inner cavity. Due to the liposome structure the lead enclosed in the liposome is more easily accepted into the cell structure and the toxicity of the enclosed lead is higher in comparison to free lead ions.

Relevance of infection with human papillomavirus: the role of the p53 tumor suppressor protein and E6/E7 zinc finger proteins (Review).

Human papillomaviruses (HPV) are small circular, double-stranded DNA viruses infecting epithelial tissues. HPV types can be classified both as high-risk or low-risk. Of the more than 120 different identified types of HPV, the majority are involved in infections of the genital tract, cancer of the cervix, vulva, vagina and penis, and of non-anogenital localizations, such as the head and neck areas. From the point of view of the infection, human papillomaviruses have developed several molecular mechanisms to enable infected cells to suppress apoptosis. This review provides a comprehensive and critical summary of the current literature that focuses on cervical carcinoma and cancer of the head and neck caused by HPV. In particular, we discuss HPV virology, the molecular mechanisms of carcinogenesis, the role of the tumor suppressor protein p53 and the E6/E7 zinc finger proteins. Classification of HPV according to diagnosis is also described.

Complexes of silver(I) ions and silver phosphate nanoparticles with hyaluronic acid and/or chitosan as promising antimicrobial agents for vascular grafts.

Polymers are currently widely used to replace a variety of natural materials with respect to their favourable physical and chemical properties, and due to their economic advantage. One of the most important branches of application of polymers is the production of different products for medical use. In this case, it is necessary to face a significant disadvantage of polymer products due to possible and very common colonization of the surface by various microorganisms that can pose a potential danger to the patient. One of the possible solutions is to prepare polymer with antibacterial/antimicrobial properties that is resistant to bacterial colonization. The aim of this study was to contribute to the development of antimicrobial polymeric material ideal for covering vascular implants with subsequent use in transplant surgery. Therefore, the complexes of polymeric substances (hyaluronic acid and chitosan) with silver nitrate or silver phosphate nanoparticles were created, and their effects on gram-positive bacterial culture of Staphylococcus aureus were monitored. Stages of formation of complexes of silver nitrate and silver phosphate nanoparticles with polymeric compounds were characterized using electrochemical and spectrophotometric methods. Furthermore, the antimicrobial activity of complexes was determined using the methods of determination of growth curves and zones of inhibition. The results of this study revealed that the complex of chitosan, with silver phosphate nanoparticles, was the most suitable in order to have an antibacterial effect on bacterial culture of Staphylococcus aureus. Formation of this complex was under way at low concentrations of chitosan. The results of electrochemical determination corresponded with the results of spectrophotometric methods and verified good interaction and formation of the complex. The complex has an outstanding antibacterial effect and this effect was of several orders higher compared to other investigated complexes.