Exploring the Molecular Mechanisms of Macrolide Resistance in Laboratory Mutant Helicobacter pylori.

Resistance to clarithromycin, a macrolide antibiotic used in the first-line treatment of Helicobacter pylori infection, is the most important cause of treatment failure. Although most cases of clarithromycin resistance in H. pylori are associated with point mutations in 23S ribosomal RNA (rRNA), the relationships of other mutations with resistance remain unclear. We examined possible new macrolide resistance mechanisms in resistant strains using next-generation sequencing. Two resistant strains were obtained from clarithromycin-susceptible H. pylori following exposure to low clarithromycin concentrations using the agar dilution method. Sanger sequencing and whole-genome sequencing were performed to detect resistance-related mutations. Both strains carried the A2142G mutation in 23S rRNA. Candidate mutations (T1495A, T1494A, T1490A, T1476A, and G1472T) for clarithromycin resistance were detected in the Mutant-1 strain. Furthermore, a novel mutation in the gene encoding for the sulfite exporter TauE/SafE family protein was considered to be linked to clarithromycin resistance or cross-resistance, being identified as a target for further investigations. In the Mutant-2 strain, a novel mutation in the gene that encodes DUF874 family protein that can be considered as relevant with antibiotic resistance was detected. These mutations were revealed in the H. pylori genome for the first time, emphasizing their potential as targets for advanced studies.

A novel rapid bioluminescence-based antimicrobial susceptibility testing method based on adenosine triphosphate consumption.

Introduction: Standard, phenotypic antimicrobial susceptibility testing (AST) methods require 16-20 h of incubation and are considered as the bottleneck in providing timely input for appropriate antimicrobial treatment. In this study, a novel adenosine triphosphate (ATP)-bioluminescence-based method which allows rapid AST within 3 h was described. Methods: Standard AST was performed for 56 Enterobacterales isolates using EUCAST disk diffusion (DD) methodology. For the bioluminescence-based rapid AST, suspensions of bacteria were prepared using Mueller-Hinton broth to obtain a turbidity of 0.5 McFarland. The suspensions were distributed into 96-well microtiter plates. ATP (20 mM) and fixed concentrations of different antibiotics were added. Following incubation at 37°C for 1 h, a luminescent reaction mixture, including the substrate luciferin and luciferase enzyme solutions, was added. The chemiluminescence was monitored using an imaging system. Light production demonstrated the presence of ATP, indicating that the isolate was susceptible to the antibiotic in the well. Absence or decrease of light intensity, compared with the growth control well, indicated the use of ATP as an indirect measure of bacterial growth, and therefore resistance to the antibiotic in the well. Results: The novel AST method was tested using a total of 348 test wells. Concordance was achieved for 290 (83.3%) of the tests, whereas 52 (14.9%) and 6 (1.7%) tests caused minor and major errors, respectively. Discussion: In this study, a bioluminescence-based rapid AST was developed based on the consumption of ATP by bacteria. Our method's uniqueness relies on determining ATP consumption by microorganisms in the presence or absence of an antibiotic. The novel AST method described in this study lays the groundwork for obtaining rapid results, which should be considered as a proof of concept. With further optimization studies, this novel method can provide higher accuracy and be introduced into clinical practice as a routine AST method.

In vitro efficacy of different PEGylation designs on cathelicidin-like peptide with high antibacterial and antifungal activity.

Recent reports on antibiotic resistance have highlighted the need to reduce the impact of this global health issue through urgent prevention and control. The World Health Organization currently considers antibiotic resistance as one of the most dangerous threats to global health. Therefore, Antimicrobial peptides (AMPs) are promising for the development of novel antibiotic molecules due to their high antimicrobial effects, non-inducing antimicrobial resistance (AMR) properties, and broad spectrum. Hence, in this study, we developed novel antimicrobial peptide/polymer conjugates to reduce the adverse effects of TN6 (RLLRLLLRLLR) peptide. We demonstrate how our constructs function in vitro in terms of antimicrobial activity, hemolytic activity, cytotoxicity, and protease resistance. Our findings show that our molecules are effective against different types of microorganisms such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant S. aureus, vancomycin-resistant Enteroccus faecium, and Candida albicans, which are known to be pathogenic and antibiotic-resistant. Our constructs generally showed low cytotoxicity relative to the peptide in HaCaT and 3T3 cells. Especially these structures are very successful in terms of hemotoxicity. In the bacteremia model with S. aureus, the naked peptide (TN6) was hemotoxic even at 1 µg/mL, while the hemotoxicity of the conjugates was considerably lower than the peptide. Remarkably in this model, the hemolytic activity of PepC-PEG-pepC conjugate decreased 15-fold from 2.36 to 31.12 µg/mL compared to the bacteria-free 60-min treatment. This is proof that in the case of bacteremia and sepsis, the conjugates specifically direct to bacterial cell membranes rather than red blood cells. In addition, the PepC-PEG-pepC conjugate is resistant to plasma proteases. Moreover, morphological and intracellular damage of the peptide/conjugates to Escherichia coli are demonstrated in SEM and TEM images. These results suggest our molecules can be considered potential next-generation broad-spectrum antibiotic molecule/drug candidates that might be used in clinical cases such as bacteremia and sepsis.

A Novel Peptide-Based Detection of SARS-CoV-2 Antibodies.

The need for rapidly developed diagnostic tests has gained significant attention after the recent pandemic. Production of neutralizing antibodies for vaccine development or antibodies to be used in diagnostic tests usually require the usage of recombinant proteins representing the infectious agent. However, peptides that can mimic these recombinant proteins may be rapidly utilized, especially in emergencies such as the recent outbreak. Here, we report two peptides that mimic the receptor binding domain of the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and investigate their binding behavior against the corresponding human immunoglobulin G and immunoglobulin M (IgG and IgM) antibodies in a clinical sample using a quartz crystal microbalance (QCM) sensor. These peptides were immobilized on a QCM sensor surface, and their binding behavior was studied against a clinical serum sample that was previously determined to be IgG and IgM-positive. It was determined that designed peptides bind to SARS-CoV-2 antibodies in a clinical sample. These peptides might be useful for the detection of SARS-CoV-2 antibodies using different methods such as enzyme-linked immunosorbent assay (ELISA) or lateral flow assays. A similar platform might prove to be useful for the detection and development of antibodies in other infections.

Simple and low-cost antibiotic susceptibility testing for Mycobacterium tuberculosis using screen-printed electrodes.

One quarter of the global population is thought to be latently infected by Mycobacterium tuberculosis (TB) with it estimated that 1 in 10 of those people will go on to develop active disease. Due to the fact that M. tuberculosis (TB) is a disease most often associated with low- and middle-income countries, it is critical that low-cost and easy-to-use technological solutions are developed, which can have a direct impact on diagnosis and prescribing practice for TB. One area where intervention could be particularly useful is antibiotic susceptibility testing (AST). This work presents a low-cost, simple-to-use AST sensor that can detect drug susceptibility on the basis of changing RNA abundance for the typically slow-growing M. tuberculosis (TB) pathogen in 96 h using screen-printed electrodes and standard molecular biology laboratory reactionware. In order to find out the sensitivity of applied sensor platform, a different concentration (108 -103  CFU/mL) of M. tuberculosis was performed, and limit of detection and limit of quantitation were calculated as 103.82 and 1011.59  CFU/mL, respectively. The results display that it was possible to detect TB sequences and distinguish antibiotic-treated cells from untreated cells with a label-free molecular detection. These findings pave the way for the development of a comprehensive, low-cost, and simple-to-use AST system for prescribing in TB and multidrug-resistant tuberculosis.

Comparison of a novel antigen detection test with reverse transcription polymerase chain reaction assay for laboratory diagnosis of SARS-CoV-2 infection.

Molecular diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time reverse transcription polymerase chain reaction (RT-PCR) in respiratory specimens is considered the gold standard method. This method is highly sensitive and specific but it has some limitations such as being expensive and requiring special laboratory equipment and skilled personnel. RapidFor™ Antigen Rapid Test Kit is a commercially available Ag-RDT which is produced in Turkey and designed to detect the nucleocapsid antigen of SARS-CoV-2 in nasopharyngeal swab samples. The aim of this study was to evaluate the performance of this novel SARS-CoV-2 antigen detection considering the RT-PCR method as the gold standard. Four hundred forty-four nasopharyngeal swab samples which were collected from the patients who met clinical criteria of COVID-19 from ten centers in Turkey between September 2020 and February 2021 were included in the study. All the nasopharyngeal swab samples were tested for SARS-CoV-2 RNA using commercial RT-PCR kits (Bioeksen and A1 Lifesciences, Istanbul, Turkey) according to the manufacturer's instructions. Viral loads were assessed according to the cycle threshold (Ct) values. RapidFor™ SARS-CoV-2 antigen test (Vitrosens Biotechnology, Istanbul, Turkey) was used to investigate the presence of SARS-CoV-2 antigen in all samples following the manufacturer's instructions. Out of 444 nasopharyngeal swab samples tested, 346 (77.9%) were positive and 98 (22.1%) were negative for SARS-CoV-2 RNA by RTPCR. Overall sensitivity of the RapidFor™. Antigen Rapid Test Kit was 80.3% whereas specificity was found to be 87.8%. Positivity rate of rapid antigen test in samples with Ct values over 25 and below 30 was 82.7%, while it increased to 95.7% in samples 20 ≤ Ct < 25 and reached 100% in samples with Ct values below 20. RapidFor™ SARS-CoV-2 Ag test might be a good choice in the screening of symptomatic and asymptomatic patients and their contacts for taking isolation measures early, with advantages over RT-PCR as being rapid, easy and being applicable in every laboratory and even at point of care.

Colibactin possessing E. coli isolates in association with colorectal cancer and their genetic diversity among Pakistani population.

Colorectal cancer (CRC) is the third most prevalent cause of tumorigenesis and several pathogenic bacteria have been correlated with aggressive cases of cancer i.e., genotoxin (colibactin) producing Escherichia coli (E. coli). This study was designed to investigate the genetic diversity of clb+clb+ E. coli strains and their association with CRC. Pathogenic E. coli isolates from colorectal biopsies were characterized based on phylotypes, antibiotic resistance pattern, and (Enterobacterial Repetitive Intergenic Consensus Sequence-based Polymerase Chain Reaction) ERIC-PCR. Furthermore, isolates were screened for the presence of the Pks (polyketide synthase) Island specifically targeting colibactin genes A and Q. The selective clb+clb+ isolates were subjected to cytotoxicity assay using Human embryonic kidney (HEK) cell lines. We revealed that 43.47% of the cancer-associated E. coli isolates were from phylogroup B2 comparatively more pathogenic than rest while in the case of healthy controls no isolate was found from B2. Moreover, 90% were found positive for colibactin and pks (polyketide synthase) island, while none of the healthy controls were found positive for colibactin genes. All healthy and cancer-associated isolates were tested against 15 antibiotic agents, we observed that cancer-associated isolates showed a wide range of resistance from 96% against Nalidixic acid to 48% against Doxycycline. Moreover, E. coli isolates were further genotyped using ERIC-PCR, and selected clb+clb+ E. coli isolates were subjected to cytotoxicity assay. We recorded the significant cytotoxic activity of clb+clb+ E. coli phylogroup B2 isolates that might have contributed towards the progression of CRC or dysbiosis of healthy gut microbiota protecting against CRC pathogenesis. Our results revealed a significant p<0.023 association of dietary habits and hygiene p<0.001with CRC. This is the first study to report the prevalence of E. coli phylogroups and the role of colibactin most virulent phylogroup B2 among Pakistani individuals from low socioeconomic setup.

Polymeric Approach to Adjuvant System in Antibody Production against Leishmaniasis Based on Hybridoma Technology.

Background: Leishmaniasis is a zoonotic disease, which is one of the serious public health problems in the world. Nowadays, antibody production using hybridoma technology may be a correct approach in terms of sensitivity in the diagnosis of diseases such as leishmaniasis. The aim of this study was investigation of the effectiveness of different adjuvants on polyclonal antibody production against L. tropica based on hybridoma technique. Methods: Accordingly, Freund's adjuvant (1956, M. tuberculosis), as a classic adjuvant in studies, was used comparatively with the non-toxic polymeric based Polyoxidonium adjuvant. All animal immunization procedures were conducted at Bezm-i Alem University Experimental Animal Research Center. The adjuvant response was tested both in the serum sample and in the antibodies produced by the hybridomas. The antibody titers were determined with ELISA. Results: Freund's and Polyoxidonium (PO) group blood titer's increased approximately 5.5 fold compared to control after the 6th and 8th immunization. Hybridomas produced from mice immunized with PO adjuvant induced only antigen-specific antibody response and did not develop an immune response against the adjuvant. Conclusion: Adjuvant selection is very important in terms of the specificity of antibody responses of cells produced in hybridoma technology. Therefore, PO is recommended as a new adjuvant system in this study.

Simple concentration method enables the use of gargle and mouthwash instead of nasopharyngeal swab sampling for the diagnosis of COVID-19 by PCR.

Since its emergence in December 2019, SARS-CoV-2 is causing one of the most devastating pandemics in human history. Currently, the most important method for definitive diagnosis of COVID-19 is identification of SARS-CoV-2 RNA in nasopharyngeal swab samples by RT-PCR. Nasopharyngeal swab sampling is a discomforting procedure sometimes with adverse effects, which also poses a risk for infection for the personnel performing the sampling. We have developed a new method for concentrating biological samples, which enabled us to use gargle and mouthwash samples to be used in RT-PCR, for the diagnosis of COVID-19, as an alternative to nasopharyngeal swab samples. We have analyzed nasopharyngeal and gargle and mouthwash samples, before and after concentration, of 363 patients by RT-PCR for the presence of SARS-CoV-2. Among 114 patients in which SARS-CoV-2 was identified in at least one of their samples, the virus was identified in 76 (66.7%), 67 (58.8%), and 101 (88.6%) of nasopharyngeal swab, gargle, and mouthwash samples before and after concentration, respectively. When concentrated by our new method, gargle and mouthwash samples can be used instead of nasopharyngeal samples in identification of SARS-CoV-2 by RT-PCR, with the same or better sensitivity. Eliminating the need for nasopharyngeal sampling will save the patients from an invasive and painful procedure and will lower the risk of infection for the healthcare personnel taking the sample. This easy sampling procedure may decrease the workload of hospitals, shorten the turnaround time of obtaining test results, and thus enable rapid isolation of infected patients.

A rapid, ultrasensitive voltammetric biosensor for determining SARS-CoV-2 spike protein in real samples.

The ongoing coronavirus disease 2019 (COVID-19) pandemic continues to threaten public health systems all around the world. In controlling the viral outbreak, early diagnosis of COVID-19 is pivotal. This article describes a novel method of voltammetrically determining severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein with a newly designed sensor involving bovine serum albumin, SARS-CoV-2 spike antibody and a functionalised graphene oxide modified glassy carbon electrode (BSA/AB/f-GO/GCE) or screen-printed electrode (BSA/AB/f-GO/SPE). The oxidation reaction based on the antibody-antigen protein interaction was evaluated as a response to SARS-CoV-2 spike protein at -200 mV and 1430 mV with the BSA/AB/f-GO/SPE and BSA/AB/f-GO/GCE, respectively. The developed sensors, BSA/AB/f-GO/SPE and BSA/AB/f-GO/GCE, could detect 1 ag/mL of virus spike protein in synthetic, saliva and oropharyngeal swab samples in 5 min and 35 min, and both sensors demonstrated a dynamic response to the SARS-CoV-2 spike protein between 1 ag/mL and 10 fg/mL. Real-time polymerase chain reaction (RT-PCR), rapid antigen test and the proposed method were applied to saliva samples. When compared to RT-PCR, it was observed that the developed method had a 92.5% specificity and 93.3% sensitivity. Moreover, BSA/AB/f-GO/SPE sensor achieved 91.7% accuracy compared to 66.7% accuracy of rapid antigen test kit in positive samples. In view of these findings, the developed sensor provides great potential for the diagnosing of COVID-19 in real samples.

Investigation of the Effect of Channel Structure and Flow Rate on On-Chip Bacterial Lysis.

Successful lysis of cells/microorganisms is a key step in the sample preparation in fields like molecular biology, bioengineering, and biomedical engineering. This study therefore aims to investigate the lysis of bacteria on-chip and its dependence on both microfluidic channel structure and flow rate. Effects of temperature on lysis on-chip were also investigated. To perform these investigations, three different microfluidic chips were designed and produced (straight, zigzag and circular configurations), while the length of the channels were kept constant. As an exemplary case, Mycobacterium smegmatis was chosen to represent the acid-fast bacteria. Bacterial suspensions of 1.5 McFarland were injected into the chips at various flow rates (0.6- [Formula: see text]/min) either at room temperature or 50° C. In order to understand the on-chip lysis performance fully, off-chip experiments were carried out at durations which are equal to those bacteria spent in the channel from inlet to the outlet at different flow rates. We also performed COMSOL multiphysics program simulations to evaluate further the effect of the applied parameters. As a result, we found that the structure and the flow rate do not affect lysis over all in all investigated channel types, however on-chip experiments at room temperature produced more effective lysis compared to the on-chip and the off-chip samples performed at higher temperatures. Interestingly on-chip experiments at higher tempratures do not result in effective lysis.

Label-free molecular detection of antibiotic susceptibility for Mycobacterium smegmatis using a low cost electrode format.

Today, the emergence of antibiotic resistance in pathogenic bacteria is considered an important problem for society. Excessive consumption of antibiotics, long-term treatments, and inappropriate prescriptions continually increase the severity of the problem. Improving antibiotic stewardship requires improved diagnostic testing, and, therefore, in vitro antibiotic susceptibility testing is becoming increasingly important. This research details the development of an antibiotic susceptibility test for Mycobacterium smegmatis using streptomycin as antibiotics. This strain was selected because it is a member of the slow growing Mycobacterium genus and serves as a useful surrogate organism for M. tuberculosis. A commercially available and low-cost screen-printed gold electrode in combination with a specifically developed nucleic acid probe sequence for the 16SrRNA region of the mycobacterial genome was employed to monitor M. smegmatis nucleic acid sequences using the techniques of square-wave voltammetry and electrochemical impedance spectroscopy. The results show that it was possible to detect M. smegmatis sequences and distinguish antibiotic-treated cells from untreated cells with a label-free molecular detection. As a result, the in vitro antibiotic susceptibility test revealed that M. smegmatis showed sensitivity to streptomycin after a 24-H incubation, with the developed protocol representing a potential approach to determining antibiotic susceptibility more quickly and economically than current methods.

Rational guidewire use in the coronary chronic total occlusion interventions.

BACKGROUND: Procedures for coronary chronic total occlusion (CTO) are still a clinical challenge with relatively lower success rates. Recent advances in the biotechnology and introduction of CTO-dedicated guidewires have increased the procedural success rate of CTO interventions. Herein, we aimed to reveal the clinical and angiographic predictors of the crossability of the initial guidewire choice and rational guidewire usage in CTO interventions. A total of 177 patients with an indication for a coronary CTO procedure were included in this study. The use of 1-3 guidewires and crossing of the CTO lesion with the initial guidewire choice was defined as rational guidewire usage. The CTO lesions were classified according to the Japanese chronic total occlusion registry (J-CTO) and EuroCTO scores for evaluating the difficulty of the procedures. Then, a statistical analysis was performed to assess the initial guidewire choice, crossability, and contributors to rational guidewire usage. RESULTS: The mean J-CTO score was 1.42 ± 1.16, and the mean EuroCTO score was 1.44 ± 1.18. The success rate of the procedures was 90.4%. The initial guidewire choice crossed the lesion in 44.1% of the cases, in which 1-3 guidewires were used (82.1%). The crossability of the polymeric and moderate stiff tip guidewires was higher (82.1% and 64.1%, respectively), and the Pilot series was the most successful brand (36.2%). Logistic regression analysis confirmed that J-CTO score, procedural technique, guidewire type, and stiffness of the tip were the major predictors of rational guidewire usage. CONCLUSION: Our analysis showed that the use of polymeric and moderate stiff tip guidewires, particularly the Pilot brand, were associated with rational guidewire usage in easy and intermediate difficulty CTO cases.

[Diversity of Leishmania Strains Isolated from Cutaneous Leishmaniasis Patients in Turkey and its Reflection to Clinics in Mice Model]. / Türkiye'de Kutanöz Leysmanyazis Hastalarindan Elde Edilen Leishmania Izolatlarindaki Farkliliklar ve Bunlarin Fare Modeline Klinik Yansimasi.

Although asexual reproduction has been attributed to Leishmania species, genetic exchange has recently been demonstrated, which helped emerging of hybrid isolates. Situated on the crossroads between three continents, Leishmania hybrids may be present in Turkey. In Turkey, visceral leishmaniasis caused by Leishmania infantum is less common, while cutaneous leishmaniasis (CL) caused by Leishmania tropica and L.infantum could reach 2500 reported cases a year. Our aim was to investigate genetic variability of local Leishmania species and presence of hybrid Leishmania strains in Turkey. Twenty CL patients from Sanliurfa and Hatay, where only L.tropica and both L.tropica and L.infantum cause CL, respectively, were registered equally. All isolates were assessed with real-time polymerase chain reaction (Rt-PCR), isoenzyme analysis, gene sequencing, two-dimensional gel electrophoresis (2D-PAGE) and MALDI-TOF/TOFMS followed by in vivo analyses on mouse model. Identification of differentially expressed proteins was performed. These proteins were confirmed by sequence analysis. All isolates from Sanliurfa were found to be L.tropica which caused cutaneous infection in mice. However, one of 10 isolates from Hatay was found as Leishmania major which caused cutaneous infection. Five isolates were found as L.tropica with Rt-PCR and gene sequencing, one of which had one different protein from the reference L.tropica strain and caused cutaneous infection. Four of the five isolates had five different proteins compared to reference strain and caused both cutaneous and visceral infections. Remaining four isolates showed double melting curves in Rt-PCR, which were concordant with L.tropica and L.infantum. Their sequencing and isoenzyme analyses indicated them as L.infantum. They had six different proteins compared to reference L.infantum strain and caused cutaneous and visceral infections. It is concluded that the isolates with different proteins were hybrid Leishmania species. In the present study, outcomes of the proteomics, genomics, clinical manifestations and tissue tropism on animal models were evaluated together for the first time. In addition to L.tropica and L.infantum, L.major was identified as a causative agent for CL and hybrids of L.infantum/tropica were also shown to be present.

Simple Identification of Mycobacterial Species by Sequence-Specific Multiple Polymerase Chain Reactions.

Several species of mycobacteria cause infections in humans. Species identification of clinical isolates of mycobacteria is very important for the decision of treatment and in choosing the appropriate treatment regimen. We have developed a multiplex PCR method that can identify practically all known species of mycobacteria, by determination of single-nucleotide differences at a total of 13 different polymorphic regions in the genes of rRNA and hsp65, in four PCR mixes. To achieve this goal, single-nucleotide differences in these polymorphic regions were used to divide mycobacterial species into two groups, than four, eight, etc., in an algorithmic manner. It was sufficient to reach single species level by evaluating 13 polymorphic regions. Evaluation of the multiplex PCR patterns by observable real-time electrophoresis (ORTE) simplified species identification. This new method may enable easy, rapid, and cost-effective identification of all species of mycobacteria.

Rapid detection of beta-lactamase production including carbapenemase by thin layer chromatography.

OBJECTIVES: To develop a rapid and simple method that can identify the presence of ß-lactamases in clinical isolates and samples, and determine their activity on different types of ß-lactam antibiotics, including carbapenems, within one hour. METHODS: In this study, we describe a thin layer chromatography-based method for rapid detection of ß-lactamases including carbapenemases. The method relies on the examination of changes in the migration rate of ß-lactams in chromatography, due to degradation by ß-lactamase enzymes. A total of 44 isolates, 29 carbapenemase-producers and 15 non-carbapenemase-producers, were screened by this method. RESULTS: The method has proven to be able to distinguish ß-lactamases as carbapenemase or non-carbapenemase producing strains with high sensitivity in one hour. CONCLUSIONS: The method developed, provides information about the production of ß-lactamases by bacteria and ß-lactam drugs inactivated by these enzymes, including carbapenems. This new method may play an important role in guiding antimicrobial treatment, especially in critically ill patients infected bacteria producing ß-lactamases.

Oligomer based real-time detection of microorganisms producing nuclease enzymes.

In this study, we provide a method using fluorescently labeled oligonucleotides for the diagnosis of microorganisms producing nucleases in real time, while growing them in culture media. The detection of such microorganisms was possible in a short period of time, as short as 10 minutes up to a maximum of 8 hours, depending on the bacterial density. We also showed the suitability of this new method for determination of minimum inhibitory concentration (MIC) in culture media in a very short period of time, compared to conventional methods. We believe that it can make a significant contribution to gain new insights for analysis of complex materials such as clinical samples, food samples and environmental samples.

Leishmaniasis in Turkey: first clinical isolation of Leishmania major from 18 autochthonous cases of cutaneous leishmaniasis in four geographical regions.

OBJECTIVE: To report isolation of Leishmania major strains obtained from 18 Turkish autochthonous cutaneous leishmaniasis (CL) patients infected with L. major between 2011 and 2014. METHODS: Initial diagnosis relied on microscopy and culture in enriched medium, prepared by adding specific amounts of liver extract, protein and lipid sources to NNN medium. Promastigotes were then transferred to RPMI medium including 10% of foetal calf serum for mass culture. Species-specific real-time PCR targeting ITS1 region of Leishmania spp. was performed using both lesion aspiration samples and cultured promastigotes. Two of 18 isolates were identified by isoenzyme analysis in the Leishmaniasis Reference Center in Montpellier, France. Each isolate was inoculated into the footpads of six mice to observe the pathogenicity of L. major. Developing lesions were observed, and the thickening of footpads was measured weekly. RESULTS: Melting curve analyses of 18 isolates showed a peak concordant with L. major, and two of them were confirmed by isoenzyme analyses as L. major zymodeme MON103. In the mouse model, acute lesions seen on day 21 were accepted as an indication of heavy infection. Severe impairments were observed on all mouse footpads over 3 weeks, which even progressed to extremity amputation. CONCLUSION: Cutaneous leishmaniasis-causing L. major was recently identified in Adana province in southern Turkey, with PCR. Our study shows that such CL cases are not limited to Adana but currently present from western to Southeastern Anatolia, and along the Mediterranean coast. The role of small mammals, the main reservoirs of L. major in Anatolia, needs to be elucidated, as do the underlying factors that cause severe clinical manifestations in L. major infections in Turkey, contrary to the infections in neighbouring countries.

Design, Synthesis, and Molecular Docking Studies of a Conjugated Thiadiazole-Thiourea Scaffold as Antituberculosis Agents.

In view of the emergence and frequency of multidrug-resistant and extensively drug-resistant tuberculosis and consequences of acquired resistance to clinically used drugs, we undertook the design and synthesis of novel prototypes that possess the advantage of the two pharmacophores of thiourea and 1,3,4-thiadiazole in a single molecular backbone. Three compounds from our series were distinguished from the others by their promising activity profiles against Mycobacterium tuberculosis strain H37Rv. Compounds 11 and 19 were the most active representatives with minimum inhibitory concentration (MIC) values of 10.96 and 11.48 µM, respectively. Compound 15 was shown to inhibit M. tuberculosis strain H37Rv with an MIC value of 17.81 µM. Cytotoxicity results in the Vero cell line showed that these three derivatives had selectivity indices between 1.8 and 8.7. In order to rationalize the biological results of our compounds, molecular docking studies with the enoyl acyl carrier protein reductase (InhA) of M. tuberculosis were performed and compounds 11, 15, and 19 were found to have good docking scores in the range of -7.12 to -7.83 kcal/mol.

Multiplex-PCR-Based Screening and Computational Modeling of Virulence Factors and T-Cell Mediated Immunity in Helicobacter pylori Infections for Accurate Clinical Diagnosis.

The outcome of H. pylori infection is closely related with bacteria's virulence factors and host immune response. The association between T cells and H. pylori infection has been identified, but the effects of the nine major H. pylori specific virulence factors; cagA, vacA, oipA, babA, hpaA, napA, dupA, ureA, ureB on T cell response in H. pylori infected patients have not been fully elucidated. We developed a multiplex- PCR assay to detect nine H. pylori virulence genes with in a three PCR reactions. Also, the expression levels of Th1, Th17 and Treg cell specific cytokines and transcription factors were detected by using qRT-PCR assays. Furthermore, a novel expert derived model is developed to identify set of factors and rules that can distinguish the ulcer patients from gastritis patients. Within all virulence factors that we tested, we identified a correlation between the presence of napA virulence gene and ulcer disease as a first data. Additionally, a positive correlation between the H. pylori dupA virulence factor and IFN-γ, and H. pylori babA virulence factor and IL-17 was detected in gastritis and ulcer patients respectively. By using computer-based models, clinical outcomes of a patients infected with H. pylori can be predicted by screening the patient's H. pylori vacA m1/m2, ureA and cagA status and IFN-γ (Th1), IL-17 (Th17), and FOXP3 (Treg) expression levels. Herein, we report, for the first time, the relationship between H. pylori virulence factors and host immune responses for diagnostic prediction of gastric diseases using computer-based models.