Escherichia coli from Human Wounds: Analysis of Resistance to ß-Lactams and Expression of RND Efflux Pumps.

Purpose: Resistance of pathogenic strains of Escherichia coli to ß-lactams, particularly to ampicillin, is on the rise and it is attributed to intrinsic and acquired mechanisms. One important factor contributing to resistance, together with primarily resistance mechanisms, is a mutation and/or an over-expression of the intrinsic efflux pumps in the resistance-nodulation-division (RND) superfamily. Among these efflux pumps, AcrA, AcrB, TolC, and AcrD play an important role in antimicrobial co-resistance, including resistance to ß-lactams. Materials and Methods: Twelve E. coli isolates obtained from patients' wounds and the control strain of E. coli ATCC 25922 were analyzed. The phenotypic resistance of these isolates to selected ß-lactams was assessed by determination of the minimal inhibitory concentration. Additionally, the prevalence of ß-lactamase genes (blaTEM, blaCTX-M, blaSHV, and blaAmpC) was screened by PCR. Real-time qPCR was used to determine the expression of the selected efflux pumps acrA, acrB, tolC, and acrD and the repressor acrR after the exposure of E. coli to ampicillin. Results: Phenotypic resistance to ß-lactams was detected in seven isolates, mainly to ampicillin and piperacillin. This was corroborated by the presence of at least one acquired bla gene in each of these isolates. Although E. coli strains varied in the expression of RND-family efflux pumps after the ampicillin exposure, their gene expression indicated that these pumps did not play a major role in the phenotypic resistance to ampicillin. Conclusion: Each E. coli isolate displayed unique characteristics, differing in minimum inhibitory concentration (MIC) values, prevalence of acquired blaTEM and blaCTX-M genes, and expression of the RND-family pumps. This together demonstrates that these clinical isolates employed distinct intrinsic or acquired resistance pathways for their defense against ampicillin. The prevalence and spread of ampicillin resistant E. coli has to be monitored and the search for ampicillin alternatives is needed.

Zinc effects on bacteria: insights from Escherichia coli by multi-omics approach.

IMPORTANCE: A long-term exposure of bacteria to zinc oxide and zinc oxide nanoparticles leads to major alterations in bacterial morphology and physiology. These included biochemical and physiological processes promoting the emergence of strains with multi-drug resistance and virulence traits. After the removal of zinc pressure, bacterial phenotype reversed back to the original state; however, certain changes at the genomic, transcriptomic, and proteomic level remained. Why is this important? The extensive and intensive use of supplements in animal feed effects the intestinal microbiota of livestock and this may negatively impact the health of animals and people. Therefore, it is crucial to understand and monitor the impact of feed supplements on intestinal microorganisms in order to adequately assess and prevent potential health risks.

Development of New Silver Nanoparticles Suitable for Materials with Antimicrobial Properties.

Silver nanoparticles are the most important nanoparticles in connection with the antimicrobial effect. Nowadays, the green synthesis of various types of nanoparticles is rapid, effective and produce less toxic nanoparticles often with specific properties. In our experiment we have developed and described in details various types of silver nanoparticles synthesized chemically or by the green synthesis. Nine different silver nanoparticles were synthesized, three by citrate method at different pHs (8; 9; 10), four using gallic acid at alkaline pHs (10; 11), and two by green synthesis using green tea and coffee extracts, both at pH 9. Characterisation of silver nanoparticles was performed using dynamic light scattering, scanning electron microscopy, and ultraviolet-visible absorption spectroscopy. Silver nanoparticles prepared by green synthesis showed the highest antioxidant activity and also ability for quenching of free radicals. Antibacterial activity of silver nanoparticles was determined on bacterial cultures such as Staphylococcus aureus and Escherichia coli. Silver nanoparticles synthesized using green tea and coffee extracts showed the highest antibacterial activity for both bacterial strains. Minimal inhibition concentration for both strains was found to be 65 µM at each silver nanoparticle synthesized using green synthesis.

Effect of arsenic (III and V) on oxidative stress parameters in resistant and susceptible Staphylococcus aureus.

The presented study deals with the observation of properties of methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in the toxic arsenic environment and influence of arsenic on antioxidant capacity. Two forms of arsenic (As(III), As(V)) with different concentrations were used for induction of the oxidative stress in tested strains. Microbiological methods showed that the growth inhibition of MSSA was higher than that of MRSA in presence of both arsenic ions. As(III) showed 24% and 33% higher anti-microbial effects than As(V) against MSSA and MRSA respectively. A similar result was found also in the experiment of reduction of biofilm-formation. By using spectrophotometry, it was revealed that As(III) induced higher antioxidant production in both bacterial cultures. Methicillin-susceptible S. aureus produced an app. 50 mg equivalent of gallic acid (GAE/1 mg of protein) and MRSA produced an app. 15 mg of GAE/1 mg of protein. The productions of metallothionein in MSSA and MRSA were decreased up to 62.41% and 55.84% respectively in presence of As ions. Reduction of As(III) and As(V) concentrations leads to a decrease in antioxidant production and increased the formation of metallothionein. All of these changes in the results were found to be significant statistically. Taken together, these experiments proved that in comparison with MSSA, MRSA is less susceptible not only to the antimicrobial effects of antibiotics but also against effects caused by metalloids, as arsenic. Thus, it can be stated that MRSA abounds with complex defensive mechanisms, which may in the future constitute significant problem in the efficiency of antibiotics alternatives as metal ions or nanoparticles.

Platinum nanoparticles induce damage to DNA and inhibit DNA replication.

Sparsely tested group of platinum nanoparticles (PtNPs) may have a comparable effect as complex platinum compounds. The aim of this study was to observe the effect of PtNPs in in vitro amplification of DNA fragment of phage λ, on the bacterial cultures (Staphylococcus aureus), human foreskin fibroblasts and erythrocytes. In vitro synthesized PtNPs were characterized by dynamic light scattering (PtNPs size range 4.8-11.7 nm), zeta potential measurements (-15 mV at pH 7.4), X-ray fluorescence, UV/vis spectrophotometry and atomic absorption spectrometry. The PtNPs inhibited the DNA replication and affected the secondary structure of DNA at higher concentrations, which was confirmed by polymerase chain reaction, DNA sequencing and DNA denaturation experiments. Further, cisplatin (CisPt), as traditional chemotherapy agent, was used in all parallel experiments. Moreover, the encapsulation of PtNPs in liposomes (LipoPtNPs) caused an approximately 2.4x higher of DNA damage in comparison with CisPt, LipoCisPt and PtNPs. The encapsulation of PtNPs in liposomes also increased their antibacterial, cytostatic and cytotoxic effect, which was determined by the method of growth curves on S. aureus and HFF cells. In addition, both the bare and encapsulated PtNPs caused lower oxidative stress (determined by GSH/GSSG ratio) in the human erythrocytes compared to the bare and encapsulated CisPt. CisPt was used in all parallel experiments as traditional chemotherapy agent.

Selenium nanoparticles as a nutritional supplement.

Selenium is an essential trace element in the diet, required for maintenance of health and growth; however, its toxicity could cause serious damage depending on dose and chemical form. Selenium nanoparticles (SeNPs) represent what we believe to be a novel prospect for nutritional supplementation because of their lower toxicity and ability to gradually release selenium after ingestion. In this review, we discuss various forms and types of SeNPs, as well as the way they are synthesized. We also discuss absorption and bioavailability of nanoparticles within the organism. SeNPs demonstrate anticancer and antimicrobial properties that may contribute to human health, not only as dietary supplements, but also as therapeutic agents.

Comparative study on toxicity of extracellularly biosynthesized and laboratory synthesized CdTe quantum dots.

Nanobiosynthesis belongs to the most recent methods for synthesis of nanoparticles. This type of synthesis provides many advantages including the uniformity in particle shape and size. The biosynthesis has also a significant advantage regarding chemical properties of the obtained particles. In this study, we characterized the basic properties and composition of quantum dots (QDs), obtained by the extracellular biosynthesis by Escherichia coli. Furthermore, the toxicity of the biosynthesized QDs was compared to QDs prepared by microwave synthesis. The obtained results revealed the presence of cyan CdTe QDs after removal of substantial amounts of organic compounds, which stabilized the nanoparticle surface. QDs toxicity was evaluated using three cell lines Human Foreskin Fibroblast (HFF), Human Prostate Cancer cells (PC-3) and Breast Cancer cells (MCF-7) and the MTT assay. The test revealed differences in the toxicity between variants of QDs, varying about 10% in the HFF and 30% in the MCF-7 cell lines. The toxicity of the biosynthesized QDs to the PC-3 cell lines was about 35% lower in comparison with the QDs prepared by microwave synthesis.

Antibody-free detection of infectious bacteria using quantum dots-based barcode assay.

Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Klebsiella pneumoniae are the most representative bacteria causing infectious diseases. Due to the increased application of antibiotics, the bacterial resistance is growing causing severe complications. Therefore, a sensitive determination of these pathogens is crucial for effective treatment. The aim of this study was to design an effective method for multiplex detection of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Klebsiella pneumoniae taking advantage from properties of magnetic particles as well as fluorescent nanoparticles (quantum dots). The method was able to detect as low concentrations of bacteria as 102 CFU/mL using the bacteria-specific genes (fnbA, mecA and wcaG).

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.

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.

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.