Electrospun polysuccinimide scaffolds containing different salts as potential wound dressing material.

In this research, we applied electrospinning to create a two-component biodegradable polymeric scaffold containing polysuccinimide (PSI) and antibacterial salts. Antibacterial agents for therapeutical purposes mostly contain silver ions which are associated with high environmental impact and, in some cases, may cause undesired immune reactions. In our work, we prepared nanofibrous systems containing antibacterial and tissue-regenerating salts of zinc acetate or strontium nitrate in different concentrations, whose structures may be suitable for developing biomedical wound dressing systems in the future. Several experiments have been conducted to optimize the physicochemical, mechanical, and biological properties of the scaffolds developed for application as wound dressings. The scaffold systems obtained by PSI synthesis, salt addition, and fiber formation were first investigated by scanning electron microscopy. In almost all cases, different salts caused a decrease in the fiber diameter of PSI polymer-based systems (<500 nm). Fourier-transform infrared spectroscopy was applied to verify the presence of salts in the scaffolds and to determine the interaction between the salt and the polymer. Another analysis, energy-dispersive X-ray spectroscopy, was carried out to determine strontium and zinc atoms in the scaffolds. Our result showed that the salts influence the mechanical properties of the polymer scaffold, both in terms of specific load capacity and relative elongation values. According to the dissolution experiments, the whole amount of strontium nitrate was dissolved from the scaffold in 8 h; however, only 50% of the zinc acetate was dissolved. In addition, antibacterial activity tests were performed with four different bacterial strains relevant to skin surface injuries, leading to the appearance of inhibition zones around the scaffold discs in most cases. We also investigated the potential cytotoxicity of the scaffolds on human tumorous and healthy cells. Except for the ones containing zinc acetate salt, the scaffolds are not cytotoxic to either tumor or healthy cells.

Development and In-Depth Characterization of Bacteria Repellent and Bacteria Adhesive Antibody-Coated Surfaces Using Optical Waveguide Biosensing.

Bacteria repellent surfaces and antibody-based coatings for bacterial assays have shown a growing demand in the field of biosensors, and have crucial importance in the design of biomedical devices. However, in-depth investigations and comparisons of possible solutions are still missing. The optical waveguide lightmode spectroscopy (OWLS) technique offers label-free, non-invasive, in situ characterization of protein and bacterial adsorption. Moreover, it has excellent flexibility for testing various surface coatings. Here, we describe an OWLS-based method supporting the development of bacteria repellent surfaces and characterize the layer structures and affinities of different antibody-based coatings for bacterial assays. In order to test nonspecific binding blocking agents against bacteria, OWLS chips were coated with bovine serum albumin (BSA), I-block, PAcrAM-g-(PMOXA, NH2, Si), (PAcrAM-P) and PLL-g-PEG (PP) (with different coating temperatures), and subsequent Escherichia coli adhesion was monitored. We found that the best performing blocking agents could inhibit bacterial adhesion from samples with bacteria concentrations of up to 107 cells/mL. Various immobilization methods were applied to graft a wide range of selected antibodies onto the biosensor's surface. Simple physisorption, Mix&Go (AnteoBind) (MG) films, covalently immobilized protein A and avidin-biotin based surface chemistries were all fabricated and tested. The surface adsorbed mass densities of deposited antibodies were determined, and the biosensor;s kinetic data were evaluated to divine the possible orientations of the bacteria-capturing antibodies and determine the rate constants and footprints of the binding events. The development of affinity layers was supported by enzyme-linked immunosorbent assay (ELISA) measurements in order to test the bacteria binding capabilities of the antibodies. The best performance in the biosensor measurements was achieved by employing a polyclonal antibody in combination with protein A-based immobilization and PAcrAM-P blocking of nonspecific binding. Using this setting, a surface sensitivity of 70 cells/mm2 was demonstrated.

Polyisobutylene-New Opportunities for Medical Applications.

This paper presents the results of the first part of testing a novel electrospun fiber mat based on a unique macromolecule: polyisobutylene (PIB). A PIB-based compound containing zinc oxide (ZnO) was electrospun into self-supporting mats of 203.75 and 295.5 g/m2 that were investigated using a variety of techniques. The results show that the hydrophobic mats are not cytotoxic, resist fibroblast cell adhesion and biofilm formation and are comfortable and easy to breathe through for use as a mask. The mats show great promise for personal protective equipment and other applications.

Colonization Dynamics of Multidrug-Resistant Klebsiella pneumoniae Are Dictated by Microbiota-Cluster Group Behavior over Individual Antibiotic Susceptibility: A Metataxonomic Analysis.

Gastrointestinal carriage of multidrug-resistant (MDR) bacteria is one of the main risk factors for developing serious, difficult-to-treat infections. Given that there is currently no all-round solution to eliminate colonization with MDR bacteria, it is particularly important to understand the dynamic process of colonization to aid the development of novel decolonization strategies. The aim of our present study was to perform metataxonomic analyses of gut microbiota dynamics during colonization with an extended-spectrum ß-lactamase (ESBL)- and carbapenemase-producing Klebsiella pneumoniae (ECKP) strain in mice; additionally, to ascertain the effects of antibiotic administration (ampicillin, ceftazidime, and ciprofloxacin) on the establishment and elimination of ECKP intestinal colonization. We have found that the phyla Bacteroidetes and Firmicutes were most dominant in all of the treatment groups; however, Bacteroidetes was more common in the groups treated with antibiotics compared to the control group. Significant differences were observed among the different antibiotic-treated groups in beta but not alpha diversity, implying that the difference is the relative abundance of some bacterial community members. Bacteria from the Lachnospiraceae family (including Agathobacter, Anaerostipes, Lachnoclostridium 11308, Lachnospiraceae UCG-004, Lachnospiraceae NK3A20 group 11318, Lachnospiraceae NK4A136 group 11319, Roseburia, and Tyzzerella) showed an inverse relationship with the carriage rate of the ECKP strain, whereas members of Enterobacteriaceae and the ECKP strain have shown a correlational relationship. Our results suggest that the composition of the microbial community plays a primary role in the MDR-colonization rate, whereas the antibiotic susceptibility of individual MDR strains affects this process to a lesser extent. Distinct bacterial families have associated into microbial clusters, collecting taxonomically close species to produce survival benefits in the gut. These associations do not develop at random, as they may be attributed to the presence of specific metabolomic networks. A new concept should be introduced in designing future endeavors for MDR decolonization, supplemented by knowledge of the composition of the host bacterial community and the identification of bacterial clusters capable of suppressing or enhancing the invader species.

The influence of antibiotics on transitory resistome during gut colonization with CTX-M-15 and OXA-162 producing Klebsiella pneumoniae ST15.

Great efforts have been made to limit the transmission of carbapenemase-producing Enterobacteriaceae (CPE), however, the intestinal reservoir of these strains and its modulation by various antibiotics remain largely unexplored. Our aim was to assess the effects of antibiotic administration (ampicillin, ceftazidime, ciprofloxacin) on the establishment and elimination of intestinal colonization with a CTX-M-15 ESBL and OXA-162 carbapenemase producing Klebsiella pneumoniae ST15 (KP5825) in a murine (C57BL/6 male mice) model. Whole genome sequencing of KP5825 strain was performed on an Illumina MiSeq platform. Conjugation assays were carried out by broth mating method. In colonization experiments, 5 × 106 CFU of KP5825 was administered to the animals by orogastric gavage, and antibiotics were administered in their drinking water for two weeks and were changed every day. The gut colonization rates with KP5825 were assessed by cultivation and qPCR. In each of the stool samples, the gene copy number of blaOXA-162 and blaCTX-M-15 were determined by qPCR. Antibiotic concentrations in the stool were determined by high pressure liquid chromatography and a bioanalytical method. The KP5825 contained four different plasmid replicon types, namely IncFII(K), IncL, IncFIB and ColpVC. IncL (containing the blaOXA-162 resistance gene within a Tn1991.2 genetic element) and IncFII(K) (containing the blaCTX-M-15 resistance gene) plasmids were successfully conjugated. During ampicillin and ceftazidime treatments, colonization rate of KP5825 increased, while, ciprofloxacin treatments in both concentrations (0.1 g/L and 0.5 g/L) led to significantly decreased colonization rates. The gene copy number blaOXA-162 correlated with K. pneumoniae in vivo, while a major elevation was observed in the copy number of blaCTX-M-15 from the first day to the fifteenth day in the 0.5 g/L dose ceftazidime treatment group. Our results demonstrate that commonly used antibiotics may have diverse impacts on the colonization rates of intestinally-carried CPE, in addition to affecting the gene copy number of their resistance genes, thus facilitating their stable persistance and dissemination.

Multiple Benefits of Plasmid-Mediated Quinolone Resistance Determinants in Klebsiella pneumoniae ST11 High-Risk Clone and Recently Emerging ST307 Clone.

International high-risk clones of Klebsiella pneumoniae are among the most common nosocomial pathogens. Increased diversity of plasmid-encoded antimicrobial resistance genes facilitates spread of these clones causing significant therapeutic difficulties. The purpose of our study was to investigate fluoroquinolone resistance in extended-spectrum beta-lactamase (ESBL)-producing strains, including four K. pneumoniae and a single K. oxytoca, isolated from blood cultures in Hungary. Whole-genome sequencing and molecular typing including multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed in selected strains. Gene expression of plasmid-mediated quinolone resistance determinants (PMQR) was investigated by quantitative-PCR. MLST revealed that three K. pneumoniae strains belonged to ST11 and one to ST307 whereas K. oxytoca belonged to ST52. The isolates harbored different ß-lactamase genes, however, all K. pneumoniae uniformly carried bla CTX-M-15. The K. pneumoniae isolates exhibited resistance to fluoroquinolones and carried various PMQR genes namely, two ST11 strains harbored qnrB4, the ST307 strain harbored qnrB1 and all K. pneumoniae harbored oqxAB efflux pump. Levofloxacin and moxifloxacin MIC values of K. pneumoniae ST11 and ST307 clones correlated with qnr and oqxAB expression levels. The qnrA1 carrying K. oxytoca ST52 exhibited reduced susceptibility to fluoroquinolones. The maintained expression of qnr genes in parallel with chromosomal mutations indicate an additional protective role of Qnr proteins that can support dissemination of high-risk clones. During development of high-level fluoroquinolone resistance, high-risk clones retain fitness thus, enabling them for dissemination in hospital environment. Based on our knowledge this is the first report of ST307 clone in Hungary, that is emerging as a potential high-risk clone worldwide. High-level fluoroquinolone resistance in parallel with upregulated PMQR gene expression are linked to high-risk K. pneumoniae clones.

Plasmid-mediated quinolone resistance among extended spectrum beta lactase producing Enterobacteriaceae from bloodstream infections.

The purpose of this study was to determine prevalence and molecular characterization of plasmid-mediated quinolone resistance (PMQR) genes [qnrA, qnrB, qnrC, qnrD, qnrS, aac(6')-Ib-cr, qepA, and oqxAB] among extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella spp. isolates from bloodcultures in Hungary. A total of 103 isolates were tested for quinolone susceptibility by microdilution method and PMQR genes were detected by polymerase chain reaction. About 40 ESBL-producing E. coli (39%) and 50 ESBL-producing Klebsiella spp. strains (48%) were resistant to ciprofloxacin; 40 ESBL-producing E. coli (39%) and 47 ESBL-producing Klebsiella spp. strains (45%) were resistant to levofloxacin; and 88 strains including 40 ESBL-producing E. coli (39%) and 48 (47%) ESBL-producing Klebsiella spp. were resistant to moxifloxacin. Among the 103 ESBL-producing isolates, 77 (75%) isolates (30 E. coli and 47 Klebsiella spp.) harbored PMQR genes. The most commonly detected gene was aac(6')-Ib-cr (65%). The occurrence of qnrS gene was 6%. Interestingly, qnrA, qnrB, qnrC, qnrD, and qepA were not found in any isolates. Among 77 PMQR-positive isolates, 27 (35.1%) and 1 (1.3%) carried two and three different PMQR genes, respectively. Only Klebsiella spp. harbored more than one PMQR genes. Observing prevalence of PMQR genes in the last 8 years, the increasing incidence of aac(6')-Ib-cr and oqxAB can be seen. Our results highlight high frequency of PMQR genes among ESBL-producing Klebsiella pneumoniae and E. coli isolates with an increasing dynamics in Hungary.