Whole-genome sequence of a novel lytic bacteriophage infecting Clavibacter michiganensis subsp. michiganensis from Turkey.

Clavibacter michiganensis subsp. michiganensis (Cmm) is an important plant-pathogenic bacterium that causes canker and wilt diseases. Biological control of the disease with bacteriophages is an alternative to conventional methods. In this study, Phage33 infecting Cmm was characterized based on morphological and genomic properties. Morphological characteristics such as shape and size were investigated using electron microscopy. The whole genome was sequenced using the Illumina Novaseq 6000 platform and the sequence was assembled and annotated. VICTOR and VIRIDIC were used for determining the phylogeny and comparing viral genomes, respectively. Electron microscopy showed that Phage33 has an icosahedral head with a diameter of ~55 nm and a long, thin, non-contractile tail ~169 nm in length. The genome of Phage33 is 56 324 bp in size, has a GC content of 62.49 % and encodes 67 open reading frames. Thirty-seven ORFs showed high homology to functionally annotated bacteriophage proteins in the NCBI database. The remaining 30 ORFs were identified as hypothetical with unknown functions. The genome contains no antimicrobial resistance, no lysogenicity and no virulence signatures, suggesting that it is a suitable candidate for biocontrol agents. The results of a blastn search showed similarity to the previously reported Xylella phage Sano, with an average nucleotide sequence identity of 92.37 % and query coverage of 91 %. This result was verified using VICTOR and VIRIDIC analysis, and suggests that Phage33 is a new member of the genus Sanovirus under the class Caudoviricetes.

Understanding the transition to viable but non-culturable state in Escherichia coli W3110: a comprehensive analysis of potential spectrochemical biomarkers.

The viable but non-culturable (VBNC) state is considered a survival strategy employed by bacteria to endure stressful conditions, allowing them to stay alive. Bacteria in this state remain unnoticed in live cell counts as they cannot proliferate in standard culture media. VBNC cells pose a significant health risk because they retain their virulence and can revive when conditions normalize. Hence, it is crucial to develop fast, reliable, and cost-effective methods to detect bacteria in the VBNC state, particularly in the context of public health, food safety, and microbial control assessments. This research examined the biomolecular changes in Escherichia coli W3110 induced into the VBNC state in artificial seawater under three different stress conditions (temperature, metal, and antibiotic). Initially, confirmation of VBNC cells under various stresses was done using fluorescence microscopy and plate counts. Subsequently, lipid peroxidation was assessed through the TBARS assay, revealing a notable increase in peroxidation end-products in VBNC cells compared to controls. ATR-FTIR spectroscopy and chemomometrics were employed to analyze biomolecular changes, uncovering significant spectral differences in RNA, protein, and nucleic acid concentrations in VBNC cells compared to controls. Notably, RNA levels increased, while protein and nucleic acid amounts decreased. ROC analyses identified the 995 cm- 1 RNA band as a consistent marker across all studied stress conditions, suggesting its potential as a robust biomarker for detecting cells induced into the VBNC state under various stressors.

Resistance mechanism of Escherichia coli strains with different ampicillin resistance levels.

Antibiotic resistance is an important problem that threatens medical treatment. Differences in the resistance levels of microorganisms cause great difficulties in understanding the mechanisms of antibiotic resistance. Therefore, the molecular reasons underlying the differences in the level of antibiotic resistance need to be clarified. For this purpose, genomic and transcriptomic analyses were performed on three Escherichia coli strains with varying degrees of adaptive resistance to ampicillin. Whole-genome sequencing of strains with different levels of resistance detected five mutations in strains with 10-fold resistance and two additional mutations in strains with 95-fold resistance. Overall, three of the seven mutations occurred as a single base change, while the other four occurred as insertions or deletions. While it was thought that 10-fold resistance was achieved by the effect of mutations in the ftsI, marAR, and rpoC genes, it was found that 95-fold resistance was achieved by the synergistic effect of five mutations and the ampC mutation. In addition, when the general transcriptomic profiles were examined, it was found that similar transcriptomic responses were elicited in strains with different levels of resistance. This study will improve our view of resistance mechanisms in bacteria with different levels of resistance and provide the basis for our understanding of the molecular mechanism of antibiotic resistance in ampicillin-resistant E. coli strains. KEY POINTS: •The mutation of the ampC promoter may act synergistically with other mutations and lead to higher resistance. •Similar transcriptomic responses to ampicillin are induced in strains with different levels of resistance. •Low antibiotic concentrations are the steps that allow rapid achievement of high antibiotic resistance.

Characterization of a Decellularized Sheep Pulmonary Heart Valves and Analysis of Their Capability as a Xenograft Initial Matrix Material in Heart Valve Tissue Engineering.

In order to overcome the disadvantages of existing treatments in heart valve tissue engineering, decellularization studies are carried out. The main purpose of decellularization is to eliminate the immunogenicity of biologically derived grafts and to obtain a scaffold that allows recellularization while preserving the natural tissue architecture. SD and SDS are detergent derivatives frequently used in decellularization studies. The aim of our study is to decellularize the pulmonary heart valves of young Merino sheep by using low-density SDS and SD detergents together, and then to perform their detailed characterization to determine whether they are suitable for clinical studies. Pulmonary heart valves of 4-6-month-old sheep were decellularized in detergent solution for 24 h. The amount of residual DNA was measured to determine the efficiency of decellularization. Then, the effect of decellularization on the ECM by histological staining was examined. In addition, the samples were visualized by SEM to determine the surface morphologies of the scaffolds. A uniaxial tensile test was performed to examine the effect of decellularization on biomechanical properties. In vitro stability of scaffolds decellularized by collagenase treatment was determined. In addition, the cytotoxic effect of scaffolds on 3T3 cells was examined by MTT assay. The results showed DNA removal of 94% and 98% from the decellularized leaflet and pulmonary wall portions after decellularization relative to the control group. No cell nuclei were found in histological staining and it was observed that the three-layer leaflet structure was preserved. As a result of the tensile test, it was determined that there was no statistically significant difference between the control and decellularized groups in the UTS and elasticity modulus, and the biomechanical properties did not change. It was also observed that decellularized sheep pulmonary heart valves had no cytotoxic effect. In conclusion, we suggest that the pulmonary valves of decellularized young Merino sheep can be used as an initial matrix in heart valve tissue engineering studies.

Revealing the single-channel characteristics of OprD (OccAB1) porins from hospital strains of Acinetobacter baumannii.

Nowadays, reports of antimicrobial resistance (AMR) against many antibiotics are increasing because of their misapplication. With this rise, there is a serious decrease in the discovery and development of new types of antibiotics amid an increase in multi-drug resistance. Unfermented Acinetobacter baumannii from gram-negative bacteria, which is one of the main causes of nosocomial infections and multi-drug resistance, has 4 main kinds of antibiotic resistance mechanism: inactivating antibiotics by enzymes, reduced numbers of porins and changing of their target or cellular functions due to mutations, and efflux pumps. In this study, characterization of the possible mutations in OprD (OccAB1) porins from hospital strains of A. baumannii were investigated using single channel electrophysiology and compared with the standard OprD isolated from wild type ATCC 19,606. For this aim, 5 A. baumannii bacteria samples were obtained from patients infected with A. baumannii, after which OprD porins were isolated from these A. baumannii strains. OprD porins were then inserted in an artificial lipid bilayer and the current-voltage curves were obtained using electrical recordings through a pair of Ag/AgCl electrodes. We observed that each porin has a characteristic conductance and single channel recording, which then leads to differences in channel diameter. Finally, the single channel data have been compared with the gene sequences of each porin. It was interesting to find out that each porin isolated has a unique porin diameter and decreased anion selectivity compared to the wild type.

Evaluation of copper-induced biomolecular changes in different porin mutants of Escherichia coli W3110 by infrared spectroscopy.

Copper (Cu), one of the heavy metals, plays a vital role in many complex biochemical reactions as a trace element. However, it often becomes toxic when its concentration in the cell exceeds a certain level. Homeostasis of metals in the cell is primarily related to regulating metal transport into and out of the cell. Therefore, it is thought that porin proteins, which have a role in membrane permeability, may also play a role in developing Cu resistance. This study identified the differences between the molecular profiles of wild-type Escherichia coli W3110 and its seven different porin mutants exposed to Cu ions using attenuated total reflectance (ATR)-Fourier transform infrared (FTIR) spectroscopy. The results showed that the absence of porin genes elicits global changes in the structure and composition of membrane lipids and proteins, in both the absence and presence of Cu. The lack of porin genes significantly elevated the amounts of fatty acids and phospholipids. When the alterations in protein secondary structures were compared, the quantity of amide I proteins was diminished by the presence of Cu. However, the amount of amide II proteins increased in porin mutant groups independent of Cu presence or absence. The DNAs are transformed from B- and Z-form to A-form due to porin mutations and the presence of Cu ions. The lack of porin genes increased polysaccharide content independent of Cu presence. This study can help characterize Cu detoxification efficiency and guide for obtaining active living cells to be used in bioremediation.

The Determination, Monitoring, Molecular Mechanisms and Formation of Biofilm in E. coli.

Biofilms are cell assemblies embedded in an exopolysaccharide matrix formed by microorganisms of a single or many different species. This matrix in which they are embedded protects the bacteria from external influences and antimicrobial effects. The biofilm structure that microorganisms form to protect themselves from harsh environmental conditions and survive is found in nature in many different environments. These environments where biofilm formation occurs have in common that they are in contact with fluids. The gene expression of bacteria in complex biofilm differs from that of bacteria in the planktonic state. The differences in biofilm cell expression are one of the effects of community life. Means of quorum sensing, bacteria can act in coordination with each other. At the same time, while biofilm formation provides many benefits to bacteria, it has positive and negative effects in many different areas. Depending on where they occur, biofilms can cause serious health problems, contamination risks, corrosion, and heat and efficiency losses. However, they can also be used in water treatment plants, bioremediation, and energy production with microbial fuel cells. In this review, the basic steps of biofilm formation and biofilm regulation in the model organism Escherichia coli were discussed. Finally, the methods by which biofilm formation can be detected and monitored were briefly discussed.

Overview of VBNC, a survival strategy for microorganisms.

Microorganisms are exposed to a wide variety of stress factors in their natural environments. Under that stressful conditions, they move into a viable but nonculturable (VBNC) state to survive and maintain the vitality. At VBNC state, microorganisms cannot be detected by traditional laboratory methods, but they can be revived under appropriate conditions. Therefore, VBNC organisms cause serious food safety and public health problems. To date, it has been determined that more than 100 microorganism species have entered the VBNC state through many chemical and physical factors. During the last four decades, dating from the initial detection of the VBNC condition, new approaches have been developed for the induction, detection, molecular mechanisms, and resuscitation of VBNC cells. This review evaluates the current data of recent years on the inducing conditions and detection methods of the VBNC state, including with microorganisms on the VBNC state, their virulence, pathogenicity, and molecular mechanisms.

On-chip label-free impedance-based detection of antibiotic permeation.

Biosensors are analytical tools used for the analysis of biomaterial samples and provide an understanding about the biocomposition, structure, and function of biomolecules and/or biomechanisms by converting the biological response into an electrical and/or optical signal. In particular, with the rise in antibiotic resistance amongst pathogenic bacteria, the study of antibiotic activity and transport across cell membranes in the field of biosensors has been gaining widespread importance. Herein, for the rapid and label-free detection of antibiotic permeation across a membrane, a microelectrode integrated microfluidic device is presented. The integrated chip consists of polydimethylsiloxane based microfluidic channels bonded onto microelectrodes on-glass and enables us to recognize the antibiotic permeation across a membrane into the model membranes based on electrical impedance measurement, while also allowing optical monitoring. Impedance testing is label free and therefore allows the detection of both fluorescent and non-fluorescent antibiotics. As a model membrane, Giant Unilamellar Vesicles (GUVs) are used and impedance measurements were performed by a precision inductance, capacitance, and resistance metre. The measured signal recorded from the device was used to determine the change in concentration inside and outside of the GUVs. We have found that permeation of antibiotic molecules can be easily monitored over time using the proposed integrated device. The results also show a clear difference between bilayer permeation that occurs through the lipidic bilayer and porin-mediated permeation through the porin channels inserted in the lipid bilayer.

Rapid fabrication of teflon apertures by controlled high voltage pulses for formation of free standing planar lipid bilayer membrane.

Free standing artificial lipid bilayers are widely used in the study of biological pores. In these types of studies, the free standing planar lipid bilayer is formed over a micron-sized aperture consisting of either polymer such as Polytetrafluoroethylene (PTFE, Teflon) or glass. Teflon is chemically inert, has a low dielectric constant, and has a high electrical resistance which combined allow for obtaining low noise recordings. This study investigates the reproducible generation of micropores in the range of 50-100 microns in diameter in a Teflon film using a high energy discharge set-up. The discharger set-up consists of a microprocessor, a transformer, a voltage regulator, and is controlled by a computer. We compared two approaches for pore creation: single and multi-pulse methods. The results showed that the multi-pulse method produced narrower aperture size distributions and is more convenient for lipid bilayer formation, and thus would have a higher success rate than the single-pulse method. The bilayer stability experiments showed that the lipid bilayer lasts for more than 33 h. Finally, as a proof-of-concept, we show that the single and multi-channel electrophysiology experiments were successfully performed with the apertures created by using the mentioned discharger. In conclusion, the described discharger provides reproducible Teflon-pores in a cheap and easy-to-operate manner.

The role of oxidative stress genes and effect of pH on methylene blue sensitized photooxidation of Escherichia coli.

In this study, the survival time of wild type E. coli W3110 and 11 mutants was analysed with a plate count method in methylene blue added or control groups under daylight fluoroscence illumination (4950 lux) at different pH values (5.0, 6.0, 7.0, and 8.0) in phosphate buffer. As a result, while the number of bacteria did not decrease under photooxidative stress at pH 5.0 and 6.0 during a 6-hour incubation, the wild type and all mutants decreased more than 2 log. at pH 8.0, and approximately one log. at pH 7.0. It was determined that a 2 log decrease in wild type E. coli takes 3.7 h according to t99 value at pH 8, these values were 2.39 h in the katE mutant, 2.64 h in the soxR mutant, 2.67 h in the oxyR mutant, 2.71 h in the sodB mutant, 3 h in the btuE mutant, 3.38 h in the zwf mutant and 3.40 h in the soxS mutant, respectively (p < 0.05). The roles of these genes were proved with complement tests. Finally, it is found that the effectiveness of photooxidative stress is in direct relation with pH, and the katE, soxR, oxyR, sodB, btuE, zwf, and soxS genes are important for the protection against this stress.

The effect of some boron derivatives on kanamycin resistance and survival of E. coli and P. aeruginosa in lake water.

OBJECTIVE: To study MIC value of 7 boron derivatives namely [Boric acid (H(3)BO(3)), Anhydrous Borax (Na(2)B(4)O(7)), Sodium Borate (NaBO(2)), Diammonium Tetraborate (NH(4))(2)B(4)O(7), Sodium Perborate (NaBO(3)), Boron Trioxide (B(2)O(3)), Potassium Tetraborate (K(2)B(4)O(7))] on E. coli and P. aeruginosa and their effects on survival of bacteria in lake water and resistance against kanamycin antibiotic. METHODS: MIC values of Boron derivatives and antibiotic were studied by broth microdilution method. The effect of boron derivatives on survival of bacteria in lake water were also determined with plate count. RESULTS: Sodium perborate was determined as the most effective substance among the studied substances. Effectiveness increased as temperature increased. E. coli was more affected from P. aeruginosa in 8 mg/mL sodium perborate concentration in lake water. Moreover, it was determined that MIC value of kanamycin antibiotic decreased 200 times by especially treating P. aeruginosa with sodium perborate in lake water. However, it can be stated that this change in resistance did not arise from microorganisms. CONCLUSION: Sodium perborate solution can be used supportedly in kanamycin antibiotic applications for P. aeruginosa. Future studies are necessary to explore the relation between sodium perborate and kanamycin which is effective on P. aeruginosa in lake water.

Screening of antioxidant, antimicrobial activities and chemical contents of edible mushrooms wildly grown in the black sea region of Turkey.

The antioxidative activity of the methanol extracts obtained from twelve commonly consumed wild edible mushrooms was investigated according to the phosphomolybdenum method, inhibition of linoleic acid peroxidation, reducing power, metal chelating, O(2)(˙-), DPPH(˙), peroxide and H(2)O(2) scavenging activity in the Black Sea Region of Turky, and compared to standard antioxidant compounds such as a-tocopherol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and trolox. Total phenolic compounds, flavonoids, anthocyanins, ascorbic acid, ß-carotene and lycopene in the extracts were measured. The antimicrobial activities of extracts were also evalated against gram (-) and (+) bacteria and yeast with disc diffusion method. Hydnum repandum, Lactarius delicious, Lactarius camphoratus, Lactarius piperatus, Ramaria flava, and Agaricus bisporus were greater than α-tocopherol and BHT on antioxidant activity. The lipid peroxidation of L. camphoratus and Lactarius volemus was excellent, and was 58.05% and 61.44% respectively. The DPPH(˙) scavenging effects decreased in the order of R. flava>BHT> Macrolepiota procera>L. camphoratus>L. piperatus>Lactarius volemus>a-tocopherol and were, at 500 µg/ml and were 80.38, 76.61, 69.74, 61.55, 61.47, 61.09, 60.65%, respectively. All the extracts of mushroom also had the stronger chelating effect according to standards. The O(2)(˙-) scavenging effect of extracts of the mushroom species was in order of R. flava>Boletus edulis>Leatiporus sulphureus>M. procera>standards. L. piperatus, L. camphorates, L. volemus, A. bisporus, Chanterellus cibarius, L. sulphureus, H. repandum showed strong antimicrobial activity, especially on E. coli. H. repandum exhibited to be more effective on P. aeruginosa than other bacterial strains.

Escherichia coli: dominance of red light over other visible light sources in establishing viable but nonculturable state.

In this study, the effect of UV-A and different wavelengths of visible light irradiations combined with or without a photosensitizer (methylene blue, MB) on the establishment of viable but nonculturable (VBNC) state in Escherichia coli was investigated. Survival of the E. coli was investigated by measuring plate counts, respiring cell count (RCC), direct viable count (DVC) and total counts over a period of up to 72 h. The inhibition rates of various light sources in the presence or absence of MB on E. coli in seawater were ranked in the order UV-A>red light>white light>blue light>green light (from greatest to least activation). E. coli survived for 10.2, 19.0, 21.3 and 24.04 h under exposure to red, white, blue and green light and for 6.8 h under exposure to UV-A in the presence of MB according to t(99). Although the VC declined to undetectable levels in a relatively short time, the RCC showed that some cells were still capable of respiration and, therefore, are assumed to have entered the VBNC phase. This is the first time that red light has been shown to have a stronger effect on E. coli survival and VBNC than white, green and blue light in seawater environment.

Viable but non-culturable state (VBNC) of Escherichia coli related to EnvZ under the effect of pH, starvation and osmotic stress in sea water.

When exposed extreme environmental conditions such as sea water, bacteria have been shown different survival strategy for continue their life. One of this strategy known as viable but nonculturable (VBNC) state which is very important for nondifferiation bacteria. VBNC cells cause serious human health problems. Little is known, however, about the genetic mechanisms underlying the VBNC state. Under different environmental conditions, porins are important in the survival strategy of bacteria. EnvZ/OmpR work together as regulators of ompF and ompC gene expression. It is known that the EnvZ system has a role in VBNC state. In this study we tried to find out the viability of EnvZ, OmpC and OmpF mutant E. coli under stress effect of osmolarity, pH and starvation. Bacteria were suspended in filtered-autoclaved sea water microcosms and numbers determined over 25 day incubation periods by plate count (PC), direct viable count (DVC) and count of cells capable of respiration (RCC). As regard to results, alkaline pH affected E. coli more than acidic pH, which led to decline in number. On the contrary glycine betaine addition to sea water protected E. coli porin mutants and also reduced the death rate of bacteria. Under the effect of pH, osmotic stress and starvation stress, wild type E. coli and porin mutants entered a dormant state or became VBNC with the exception of MSZ31 (envZ mutant) E. coli cells which did not enter the VBNC state under the three tested stress conditions. This study is the first report to demonstrate that E. coli could not enter the VBNC state in the lack of EnvZ product under the stress of osmolarity, pH and starvation and the relationship between EnvZ and VBNC state are not affected by pH, osmolarity and starvation.