A metagenomic survey of bacterial communities from kurut: The fermented cow milk in Kyrgyzstan.

Kurut is a traditional dry dairy product mostly consumed in Central Asia. In this study, the distribution of the dominant bacteria present in kurut samples (n=84) originated from seven (Chuy, Issyk-Kul, Talas, Naryn, Jalal-Abad, Osh, and Batken) regions in Kyrgyzstan were analyzed with Illumina iSeq100 platform. The dominant phylum detected was Firmicutes followed by Proteobacteria, Actinobacteria, Cyanobacteria/Chloroplast, and Tenericutes. The most abundant family detected was Lactobacillaceae followed by Streptococcaceae, Enterococcaceae, Chloroplast, and Leuconostocaceae. At the genus level, Lactobacillus was the predominant one in samples and Streptococcus, Enterococcus, Lactococcus, and Streptophyta followed this. Further comprehensive characterization analyses in kurut samples may have potential applications both in industrial starter culture developments and also future therapeutic approaches based on potential strains with probiotic properties.

Direct Detection of Viral Infections from Swab Samples by Probe-Gated Silica Nanoparticle-Based Lateral Flow Assay.

Point-of-care diagnosis is crucial to control the spreading of viral infections. Here, universal-modifiable probe-gated silica nanoparticles (SNPs) based lateral flow assay (LFA) is developed in the interest of the rapid and early detection of viral infections. The most superior advantage of the rapid assay is its utility in detecting various sides of the virus directly from the human swab samples and its adaptability to detect various types of viruses. For this purpose, a high concentration of fluorescein and rhodamine B as a reporting material was loaded into SNPs with excellent loading capacity and measured using standard curve, 4.19 µmol ⋅ g-1 and 1.23 µmol ⋅ g-1 , respectively. As a model organism, severe acute respiratory syndrome coronavirus-2 (CoV-2) infections were selected by targeting its nonstructural (NSP9, NSP12) and envelope (E) genes as target sites of the virus. We showed that NSP12-gated SNPs-based LFA significantly outperformed detection of viral infection in 15 minutes from 0.73 pg ⋅ mL-1 synthetic viral solution and with a dilution of 1 : 103 of unprocessed human samples with an increasing test line intensity compared to steady state (n=12). Compared to the RT-qPCR method, the sensitivity, specificity, and accuracy of NSP12-gated SNPs were calculated as 100 %, 83 %, and 92 %, respectively. Finally, this modifiable nanoparticle system is a high-performance sensing technique that could take advantage of upcoming point-of-care testing markets for viral infection detections.

The Effects of Paddy Cultivation and Microbiota Members on Arsenic Accumulation in Rice Grain.

Access to safe food is one of the most important issues. In this context, rice plays a prominent role. Because high levels of arsenic in rice grain are a potential concern for human health, in this study, we determined the amounts of arsenic in water and soil used in the rice development stage, changes in the arsC and mcrA genes using qRT-PCR, and the abundance and diversity (with metabarcoding) of the dominant microbiota. When the rice grain and husk samples were evaluated in terms of arsenic accumulation, the highest values (1.62 ppm) were obtained from areas where groundwater was used as irrigation water, whereas the lowest values (0.21 ppm) occurred in samples from the stream. It was observed that the abundance of the Comamonadaceae family and Limnohabitans genus members was at the highest level in groundwater during grain formation. As rice development progressed, arsenic accumulated in the roots, shoots, and rice grain. Although the highest arsC values were reached in the field where groundwater was used, methane production increased in areas where surface water sources were used. In order to provide arsenic-free rice consumption, the preferred soil, water source, microbiota members, rice type, and anthropogenic inputs for use on agricultural land should be evaluated rigorously.

Bacterial Skin Microbiota of Seabass from Aegean Fish Farms and Antibiotic Susceptibility of Psychrotrophic Pseudomonas.

Farming seabass (Dicentrarchus labrax) is an essential activity in the Mediterranean basin including the Aegean Sea. The main seabass producer is Turkey accounting for 155,151 tons of production in 2021. In this study, skin swabs of seabass farmed in the Aegean Sea were analysed with regard to the isolation and identification of Pseudomonas. Bacterial microbiota of skin samples (n = 96) from 12 fish farms were investigated using next-generation sequencing (NGS) and metabarcoding analysis. The results demonstrated that Proteobacteria was the dominant bacterial phylum in all samples. At the species level, Pseudomonas lundensis was identified in all samples. Pseudomonas, Shewanella, and Flavobacterium were identified using conventional methods and a total of 46 viable (48% of all NGS+) Pseudomonas were isolated in seabass swab samples. Additionally, antibiotic susceptibility was determined according to standards of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) in psychrotrophic Pseudomonas. Pseudomonas strains were tested for susceptibility to 11 antibiotics (piperacillin-tazobactam, gentamicin, tobramycin, amikacin, doripenem, meropenem, imipenem, levofloxacin, ciprofloxacin, norfloxacin, and tetracycline) from five different groups of antibiotics (penicillins, aminoglycosides, carbapenems, fluoroquinolones, and tetracyclines). The antibiotics chosen were not specifically linked to usage by the aquaculture industry. According to the EUCAST and CLSI, three and two Pseudomonas strains were found to be resistant to doripenem and imipenem (E-test), respectively. All strains were susceptible to piperacillin-tazobactam, amikacin, levofloxacin, and tetracycline. Our data provide insight into different bacteria that are prevalent in the skin microbiota of seabass sampled from the Aegean Sea in Turkey, and into the antibiotic resistance of psychrotrophic Pseudomonas spp.

Detection of viruses by probe-gated silica nanoparticles directly from swab samples.

Viral infection has been one of the major health issues for human life. The real-time reverse transcription polymerase chain reaction (RT-PCR)-based detection has primarily been used for virus detection as a highly reliable procedure. However, it is a relatively long and multi-stage process. In addition, required skilled personnel and complex instrumentation presents difficulties in large scale monitoring efforts. Therefore, we report here a direct and fast detection method for CoV-2 genome as applied in the nose-throat swab samples without any further processing. The detection principle is based on fluorescein-loaded mesoporous silica nanoparticles capped by specific gene sequences probes immobilized on the surface of the nanoparticles. Upon hybridization with the target viral genome, the fluorescein molecules were released from the mesopores. Testing with synthetic oligonucleotides, the NSP12 gene-based detection resulted in a strong signal. Target detection time could be optimized to 15 min and the limit of detection was 1.4 RFU with 84% sensitivity with clinical samples (n = 43).

Surface microbiota and associated staphylococci of houseflies (Musca domestica) collected from different environmental sources.

Houseflies (Musca domestica) are important mechanical vectors for the transmission of pathogenic microorganisms. In this study, 129 houseflies (69 males and 60 females) were collected from 10 different environmental sources and a laboratory population was used. The surface microbiota of houseflies was identified by Next-Generation Sequencing. Staphylococci from the surfaces of houseflies were selectively isolated and their virulence genes, antibiotic susceptibilities, biofilm formation, and clonal relatedness were determined. Metagenomic analysis results demonstrated that Staphylococcus, Bacillus, and Enterococcus were mostly present on the surface of houseflies at the genus level. Additionally, the isolated 32 staphylococcal strains were identified as Staphylococcus sciuri (n = 11), S. saprophyticus (n = 9), S. arlettae (n = 6), S. xylosus (n = 4), S. epidermidis (n = 1) and S. gallinarum (n = 1). tetK, tetM, tetL, ermC, msrAB, and aad6 genes were found to carry by some of the staphylococcal strains. The strains were mostly resistant to oxacillin, penicillin, and erythromycin and three strains were multi-drug resistant. There was a statistical difference between housefly collection places and antibiotic resistance of isolated staphylococci to penicillin G, gentamicin, and erythromycin (p < 0.05). Biofilm test showed that 17 strains were strong biofilm formers, and it plays important role in the transmission of these bacteria on the surface of houseflies. Staphylococcal strains showed extracellular proteolytic and lipolytic activity in 31 and 12 strains, respectively. Closely related species were found in PFGE analysis from different environmental sources. By this study, surface microbiota and carriage of pathogenic staphylococci on the surfaces of houseflies and their virulence properties were elucidated.

Development of electrochemical aptasensors detecting phosphate ions on TMB substrate with epoxy-based mesoporous silica nanoparticles.

This study, it is aimed to develop an electrochemical aptasensor that can detect phosphate ions using 3.3'5.5' tetramethylbenzidine (TMB). It is based on the principle of converting the binding affinity of the target molecule phosphate ion (PO43-) into an electrochemical signal with specific aptamer sequences for the aptasensor to be developed. The aptamer structure served as a gate for the TMB to be released and was used to trap the TMB molecule in mesoporous silica nanoparticles (MSNPs). The samples for this study were characterized by transmission electron spectroscopy (TEM), Brunner-Emmet-Teller, dynamic light scattering&electrophoretic light scattering, and induction coupled plasma atomic emission spectroscopy. According to TEM analysis, MSNPs have a morphologically hexagonal structure and an average size of 208 nm. In this study, palladium-carbon nanoparticles (Pd/C NPs) with catalytic reaction were used as an alternative to the biologically used horseradish peroxidase (HRP) enzyme for the release of TMB in the presence of phosphate ions. The limit of detection (LOD) was calculated as 0.983 µM, the limit of determination (LOQ) was calculated as 3.276 µM, and the dynamic linear phosphate range was found to be 50-1000 µM. The most important advantage of this bio-based aptasensor assembly is that it does not contain molecules such as a protein that cannot be stored for a long time at room temperature, so its shelf life is very long compared to similar systems developed with antibodies. The proposed sensor shows good recovery in phosphate ion detection and is considered to have great potential among electrochemical sensors.

High-efficiency application of CTS-Co NPs mimicking peroxidase enzyme on TMB(ox).

In this study, analytical studies of Chitosan-Cobalt(II) (CTS-Co(II)) nanoparticles (CTS - Co NPs) by mimicking horseradish peroxidase (HRP) were evaluated. In the applications, it was observed that CTS-Co NPs 3,3' 5,5' tetramethylbenzidine (TMB) oxidized in the presence of hydrogen peroxide (H2O2). The required CTS-Co NPs were synthesized at 50 °C in 30 min and characterized using Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), inductively coupled plasma-optical emission spectroscopy (ICP-OES), and X-ray photon spectroscopy (XPS) was done. CTS-Co NPs were studied to develop a selective TMB biosensor on TMB(ox) substrate. The synthesized CTS-Co NPs formed a catalytic reaction with 30% 0.2 mM H2O2 on 0.2 M TMB substrate. After the catalytic reaction, sensitive signals were obtained from the desired biosensor. Electrochemical measurements were taken as low limit of 10 mg and a high limit of 20 mg for the determination of CTS-Co NPs to TMB(ox). In the microplate study; The sensors were applied on 1.5 µg and 3 µg CTS-Co NPs TMB(ox) substrate, respectively. CTS- Co NPs; for TMB(ox) determination, optical density (OD) measurement was taken as a low limit of 1.5 µg and a high limit of 3 µg. Electrochemical applications of particles and microplate reader results were compared with horseradish peroxidase (HRP) enzyme for sensor properties. According to the data obtained, it was observed that it behaved similarly to the CTS-Co NPs peroxidase enzyme. This work presents innovations for nanoparticle extraction and sensor study from chitosan and other naturally sourced polymers.

Microbial community of soda Lake Van as obtained from direct and enriched water, sediment and fish samples.

Soda lakes are saline and alkaline ecosystems that are considered to have existed since the first geological records of the world. These lakes support the growth of ecologically and economically important microorganisms due to their unique geochemistry. Microbiota members of lakes are valuable models to study the link between community structure and abiotic parameters such as pH and salinity. Lake Van is the largest endroheic lake and in this study, bacterial diversity of lake water, sediment, and pearl mullet (inci kefali; Alburnus tarichi), an endemic species of fish which are collected from different points of the lake, are studied directly and investigated meticulously using a metabarcoding approach after pre-enrichment. Bacterial community structures were identified using Next Generation Sequencing of the 16S rRNA gene. The analysis revealed that the samples of Lake Van contain high level of bacterial diversity. Direct water samples were dominated by Proteobacteria, Cyanobacteria, and Bacteroidota, on the other hand, pre-enriched water samples were dominated by Proteobacteria and Firmicutes at phylum-level. In direct sediment samples Proteobacteria, whereas in pre-enriched sediment samples Firmicutes and Proteobacteria were determined at highest level. Pre-enriched fish samples were dominated by Proteobacteria and Firmicutes at phylum-level. In this study, microbiota members of Lake Van were identified by taxonomic analysis.

Nuclease activity as a specific biomarker for breast cancer.

We report on the activity of nucleases derived from cancer cells as a means for specific targeting using nucleic acid probes (substrates). We hypothesize that cancer cells can be differentiated from healthy cells based on their nuclease activity profile, and thus, any method based on this property represents a novel alternative for diagnostic and therapeutic intervention.

[A sepsis case caused by a rare opportunistic pathogen: Bacillus pumilus]. / Nadir görülen firsatçi patojen Bacillus pumilus'un neden oldugu bir sepsis olgusu.

The high prevalence of Bacillus species in nature and the detection of these bacteria as contaminant in cultures may lead diagnostic dilemma, however they should still be considered as a pathogen particularly in case of repeated positive cultures from patients with risk factors. Bacillus pumilus is a bacteria, though rarely, been reported as the causative agent of various infections such as sepsis, endocarditis, skin infections and food poisoning in human. In this report, a sepsis case in an immunocompetent patient caused by B.pumilus was presented. A 38-year-old female patient was admitted to emergency service of our hospital with the complaints of headache, dizziness and diarrhea. She had not any risk factors except a history of heart valve replacement operation two years ago. In physical examination, she had abdominal retention, high fever and hypotension, together with the high levels of sedimentation rate (ESR) and C-reactive protein (CRP). The patient was hospitalized with the preliminary diagnosis of sepsis. Three sets of blood samples at two different periods were taken for the culture. All blood culture vials had a positive signal at the second day of incubation in BD BACTEC™ 9050 system, therefore subcultures were performed in sheep blood agar, chocolate agar and MacConkey agar, and incubated in aerobic and anaerobic conditions. Beta-haemolytic, gray-colored large colonies were isolated from anaerobic culture at the end of 18-24 hours incubation, and Gram staining from colonies showed gram-positive rods. The isolate was identified as B.pumilus with 99% accuracy rate by using BD Phoenix™ 100 identification system. This result was also confirmed by MALDI-TOF based VITEK® MS system and 16S rRNA sequencing by Illumina MiSeq® platform. Antibiotic susceptibility test performed by BD Phoenix™ 100 system and the isolate was found to be resistant against penicillin, while it was susceptible to vancomycin, erythromycin, clindamycin, levofloxacin, and trimethoprim-sulfamethoxazole. Initial treatment of patient was started with intravenous ceftriaxone and metronidazole empirically. Hypotension and fever returned to normal levels at the second and third days of the treatment, respectively. Metronidazole treatment was stopped at seventh day, and treatment was completed to 14 day with ceftriaxone alone. At the end of the treatment course, general condition of the patient was completely good, ESR and CRP were also decreased to normal levels. In conclusion, although most of the reported bloodstream infections that are caused by B.pumilus are intravascular catheter-related, artificial heart valves should also be considered as a risk factor even though vegetation was not detected in our patient.

Selection of Aptamers for Metabolite Sensing and Construction of Optical Nanosensors.

Optical nanosensors are based on particles with diameters from 20 to 200 nm containing sensory elements. The latter are comprised of one or more signaling molecules and one or more references, which allow measurements to be ratiometric and hence independent on the amount of sensor. The signaling molecules may range from simple ion-binding fluorophores, e.g., pH-sensitive dyes, to complex biochemical assays. Aptamers are ideal for use in nanosensors because they are relatively easy to modify chemically and hence to transform into signaling molecules, and their binding affinities may be fine-tuned to a desired measuring range in the selection process. Here we first describe the selection of metabolite binding aptamers, how they are transformed into signaling molecules using a molecular beacon construct and then how they are inserted into nanoparticles. Finally, we briefly describe how the sensors are calibrated before inserted into cells to measure metabolite concentration in real time. As examples we present aptamers binding to key metabolites in cells: ATP and fructose 1, 6-bisphosphate (FBP).

Comparison of a Novel Test (ODAK Brucella Coombs Gel Test) with Commonly Used Serological Tests in Human Brucellosis.

BACKGROUND: Since Brucellosis is difficult to diagnose based on clinical symptoms, the diagnosis mostly relies on the results of serological testing. ODAK Brucella Coombs Gel Test is a novel and rapid gel microcolumn agglutination test which is performed in microcolumns containing gel matrix and Coombs antibodies. In this study, we aimed to compare ODAK Brucella Coombs Gel Test with other commonly used serological tests. METHODS: 150 blood samples of patients, preliminarily diagnosed as Brucellosis, were included in this study. Rose Bengal (RB), ODAK Brucella Coombs Gel Test (CGT), Brucellacapt (BCAP), and Standard Agglutination Test (SAT) were performed for all samples. Also, Coombs Agglutination Test (CAT) was performed for all SAT negative samples. 1/160 and above titers were accepted as positive result except RB which is a qualitative test. RESULTS: 100 (67%) out of 150 samples were found positive by RB. All of the 50 RB negative samples were also found negative by SAT and CAT test. However, 2 (4%) and 7 (14%) of them were positive by CGT and BCAP tests, respectively. Additionally, among 100 RB positive samples, only 68, 77, and 87 were positive by SAT+CAT combination, CGT, and BCAP tests, respectively. CONCLUSIONS: Currently, CGT is the only rapid (< 1 hour) serological test in which Coombs antibodies are used. Our results showed that negative results of RB, as a screening test, are not reliable enough as compared to CGT. However, positive RBT results confirmed with SAT were almost always, in most of the cases with higher titers, positive with CGT and BCAP. On the other hand, even if SAT is found negative with RB positivity, samples still must be investigated with CAT, CGT or BCAP. Consequently, CGT may be used as a rapid screening test instead of RB and it furthermore has similar sensitivity with the other confirmation tests in which Coombs antibodies are used. Therefore, ODAK Brucella Coombs Gel Test seems to be a very useful diagnostic tool for Brucellosis.

Aptamers: molecular tools for medical diagnosis.

Aptamers have been increasingly applied in biomedical field as a class of biorecognition elements that possess many advantages such as high specificity and binding affinity, easy synthesis, easy modification, small size, non-toxicity and good stability. Many diseases like cancer exhibit cellular aberrations at morphological and molecular levels. Medical diagnosis based on molecular features can be highly specific and extremely sensitive when proper recognition molecule and an efficient signal transduction system are employed. However, bioanalysis of human diseases at the molecular level is an extremely challenging field because effective probes to identify and recognize biomarkers of diseases are not readily available. Traditional bio-recognition molecule, antibody has been exploited to develop excellent diagnosis assays in many formats, but antibodies are insufficient to match the requirements of fast and portable biosensors for point-of-care applications, which are at high demand in pathogenic bacteria detection as well as other diseases like cancer. Aptamers are short single-stranded oligonucleotides, which can be selected from random combinatorial library by SELEX in vitro. This relatively new biorecognition agent has superior intrinsic characteristics for biosensor development. In this review, we first present major aptamer selection technologies and the main formats of biosensors, which were frequently employed in aptasensor development. Then, the current state of aptamers as applied to medical diagnosis was discussed for specifically cancer and pathogen diagnosis. Finally, an overview of aptamer-nanomaterials conjugates was presented in many applications such as diagnosis, bioimaging, and theranostics.

DNA-aptamer gating membranes.

This report describes a membrane barrier whose permeability is modulated through the recognition of a small-molecule target, adenosine triphosphate (ATP), by a DNA-aptamer. The gating function of the DNA-aptamer in the stimulus-responsive membrane was shown to be specific, concentration dependent, and reversible.

DNA aptamers are functional molecular recognition sensors in protic ionic liquids.

The function and structural changes of an AMP molecular aptamer beacon and its molecular recognition capacity for its target, adenosine monophosphate (AMP), was systematically explored in solution with a protic ionic liquid, ethylammonium nitrate (EAN). It could be proven that up to 2 M of EAN in TBS buffer, the AMP molecular aptamer beacon was still capable of recognizing AMP while also maintaining its specificity. The specificity was proven by using the guanosine monophosphate (GMP) as target; GMP is structurally similar to AMP but was not recognized by the aptamer. We also found that in highly concentrated EAN solutions the overall amount of double stranded DNA formed, as well as its respective thermal stability, diminished gradually, but surprisingly the hybridization rate (kh ) of single stranded DNA was significantly accelerated in the presence of EAN. The latter may have important implications in DNA technology for the design of biosensing and DNA-based nanodevices in nonconventional solvents, such as ionic liquids.

Portable bioactive paper-based sensor for quantification of pesticides.

A paper-based biosensor was developed for the detection of the degradation products of organophosphorus pesticides. The biosensor quantifies acetylcholine esterase inhibitors in a fast, disposable, cheap, and accurate format. We specifically focused on the use of sugar or protein stabilizer to achieve a biosensor with long shelf-life. The new biosensor detected malathion with a detection limit of 2.5 ppm in 5 min incubation time. The operational stability was confirmed by testing 60 days storage at 4°C when glucose was used as stabilizer.

Antimicrobial aptamers for detection and inhibition of microbial pathogen growth.

Discovery of alternative sources of antimicrobial agents are essential in the ongoing battle against microbial pathogens. Legislative and scientific challenges considerably hinder the discovery and use of new antimicrobial drugs, and new approaches are in urgent demand. On the other hand, rapid, specific and sensitive detection of airborne pathogens is becoming increasingly critical for public health. In this respect affinity oligonucleotides, aptamers, provide unique opportunities for the development of nanotechnological solutions for such medical applications. In recent years, aptamers specifically recognizing microbial cells and viruses showed great potential in a range of analytical and therapeutic applications. This article describes the significant advances in the development of aptamers targeting specific pathogens. Therapeutic application of aptamers as neutralizing agents demonstrates great potential as a future source of antimicrobial agent.

Dual-polarization interferometry for quantification of small molecules using aptamers.

An interferometry-based method was developed for detection of a small molecule, argininamide. The quantification of argininamide was demonstrated using aptamers immobilized on silicone oxynitride sensor surface via avidin-biotin binding. The aptamers formed a thin film over avidin layer corresponding to a thickness of 1.2 nm, consistent with a molecular positioning of multipoint attachment to the surface. The binding of argininamide did not cause any significant changes in the thickness of the aptamer film, suggesting that the specific binding did not affect the overall conformation of the aptamer molecules after adaptive rearrangement of aptamer molecules. However, the binding results in clearly detectable changes in mass calculated from multiple parameters determined by mass deposition and structural changes. The limit of detection of the developed sensor was determined to be 5 µM. The sensor can monitor real-time changes in argininamide concentrations with high reliability and sensitivity. The model system demonstrated that a combined measurement considering structural and mass changes through interferometry-based techniques can overcome one of the major problems associated with real-time monitoring of small mass analytes.

Measurements of intracellular ATP provide new insight into the regulation of glycolysis in the yeast Saccharomyces cerevisiae.

Glycolysis in the yeast Saccharomyces cerevisiae exhibits temporal oscillation under anaerobic or semianaerobic conditions. Previous evidence indicated that at least two membrane-bound ATPases, the mitochondrial F(0)F(1) ATPase and the plasma membrane P-type ATPase (Pma1p), were important in regulating the glycolytic oscillation. Measurements of intracellular ATP provide a unique tool to understand the role of these membrane ATPases and how their activities are regulated. We have constructed a new nanobiosensor that can perform time-resolved measurements of intracellular ATP in intact cells. Measurements of the temporal behaviour of intracellular ATP in a yeast strain with oscillating glycolysis showed that, in addition to oscillation in intracellular ATP, there is an overall slow decrease in intracellular ATP because the ATP consumption rate exceeds the ATP production in glycolysis. Measurements of the temporal behaviour of intracellular ATP in yeast strains lacking either of the two membrane bound ATPases have confirmed that F(0)F(1) ATPase and Pma1p contribute significantly to the ATP consumption in the cell and to the regulation of glycolytic oscillation. Furthermore, our measurements also demonstrate that ATPase activity is under strict control. In the absence of glucose ATPase activity is switched off, and the intracellular ATP concentration is high. When glucose is added to the cells the ATP concentration starts to decrease, because ATP consumption exceeds ATP production by glycolysis. Finally, when glucose is used up, the ATP consumption stops immediately. Thus, glucose or some compound derived from glucose must be involved in controlling the activity of these two ATPases.