Carbapenem and Colistin Resistance, Integrons and Plasmid Replicon Types in Multi-Drug Resistant Klebsiella Strains Isolated in Turkey.

BACKGROUND: Multidrug-resistant (MDR) Klebsiella is a globally important nosocomial pathogen. In the present study, 101 multidrug-resistant Klebsiella strains isolated from various clinical specimens obtained from two different Medical Faculties' hospitals were involved. We aimed to find out the prevalence of carbapenemase, mobile colistin resistance genes, and integrons in MDR Klebsiella strains. METHODS: The antibiotic susceptibilities of strains were determined by Kirby Bauer disc-diffusion method and resistance to colistin was confirmed by detection of minimum inhibitory concentrations. The prevalence of carba-penemase genes (blaOXA-48, blaNDM, blaIMP, blaVIM, blaKPC), mobile colistin-resistance genes (mcr-1 and mcr-2), and integrons (class I, II and III) were examined in Klebsiella strains by polymerase chain reaction. RESULTS: All strains were resistant to ß-lactam antibiotics, carbapenems, and quinolones. On the other hand, only nine (8.9%) strains were resistant to colistin. The most common carbapenemase genes were blaNDM (64.3%) and blaOXA-48 (53.5%). Besides, 28 (27.7%) strains were found to harbor both blaNDM and blaOXA-48. These 28 strains be-longed to the IncA/C (18.7%), IncL/M (7.7%), and IncFIIs (1.1%) plasmid replicon types. No strain was positive for blaIMP, mcr-1, and mcr-2. Class I and Class II integrons were shown to be harbored in 83.2% and 63.3% of strains, respectively. In total, 63 (63.6%) of strains harbored both classes I and II integrons. Class III integron was not detected. There was a statistically significant relationship between the presence of integrons and antibiotic resistance for cefotaxime (p = 0.024), ciprofloxacin (p < 0.001) trimethoprim/sulfamethoxazole (p < 0.001) and levofloxacin (p = 0.002). To our knowledge, this study represents the first report of a human isolate for the co-presence of blaNDM, blaOXA-48 and both Class I and Class II integrons, from Turkey. CONCLUSIONS: Our findings also highlight the dissemination of integrons and carbapenemases and the importance of surveillance on emerging antibiotic resistance.

Are insect GPCRs ideal next-generation pesticides: opportunities and challenges.

The increasing human population, combined with low inefficiency and adverse effects of available pesticides, has magnified the urgent need of developing next-generation pesticides. Among the available approaches, strategies targeting invertebrate G protein-coupled receptors (GPCRs) are very promising as these receptors are the targets of endogenous neuropeptides/neuromodulators that upon binding to their receptors induce profound changes in insect physiology. Therefore, exploring GPCR regulators has great potential in the development of targeted next-generation pesticides. Despite the great potential of such alternative pesticides, so far there has been only one approved compound, Amitraz, which conveys its anti-pest activity via the GPCR Octopamine receptor. Here, we review the current status of pesticide development, hazards associated with conventional pesticide compounds, alternative strategies that involve next-generation of pesticides, structural features of GPCRs, and opportunities and challenges of targeting the members of this superfamily in invertebrates to develop anti-pest agents. In conclusion, we emphasize that the potential of GPCRs cannot be utilized in full without more genomic and transcriptomic data to improve our understanding of the complex network of peptidergic signaling pathways. We argue how vital it is to obtain three-dimensional (3D) structural data on physiologically important target GPCRs and encourage the readers to use the state of the art in silico methods such as virtual screening for the discovery of new pesticide compounds.

Bacterial Shoot Apical Meristem Inoculation Assay.

By virtue of their sessile nature, plants may not show the fight-and-flight response, but they are not devoid of protecting themselves from disease-causing agents, attack by herbivores, and damages that are caused by other environmental factors. Plants differentially protect their life-sustaining organs such as plant apexes from the attack by microbial pathogens. There are well-established methods to inoculate/infect various plant parts such as leaves, roots, and stems with various different pathogens. The plant shoot apical meristems (SAM) are a high-value plant target that provides niche to stem cell populations. These stem cells are instrumental in maintaining future plant progenies by giving birth to cells that culminate in flowers, leaves, and stems. There are hardly few protocols available that allow us to study immune dynamics of the plant stem cells as they are hindered by various layers of the SAM cell populations. Here, we describe a step-by-step method on how to inoculate the Arabidopsis SAM with model plant pathogen Pseudomonas syringae pv. tomato DC3000.

Hyperthermophilic flavin reductase from Sulfolobus solfataricus P2: Production and biochemical characterization.

Nicotinamide adenine dinucleotide phosphate (NAD(P)H)-flavin oxidoreductases (flavin reductases) catalyze the reduction of flavin by NAD(P)H and provide the reduced form of flavin mononucleotide (FMN) to flavin-dependent monooxygenases. Based on bioinformatics analysis, we identified a putative flavin reductase gene, sso2055, in the genome of hyperthermophilic archaeon Sulfolobus solfataricus P2, and further cloned this target sequence into an expression vector. The cloned flavin reductase (EC. 1.5.1.30) was purified to homogeneity and characterized further. The purified enzyme exists as a monomer of 17.8 kDa, free of chromogenic cofactors. Homology modeling revealed this enzyme as a TIM barrel, which is also supported by circular dichroism measurements revealing a beta-sheet rich content. The optimal pH for SSO2055 activity was pH 6.5 in phosphate buffer and the highest activity observed was at 120 °C within the measurable temperature. We showed that this enzyme can use FMN and flavin adenine dinucleotide (FAD) as a substrate to generate their reduced forms. The purified enzyme is predicted to be a potential flavin reductase of flavin-dependent monooxygenases that could be involved in the biodesulfurization process of S. solfataricus P2.

Current progress of genetically engineered pig models for biomedical research.

The first transgenic pigs were generated for agricultural purposes about three decades ago. Since then, the micromanipulation techniques of pig oocytes and embryos expanded from pronuclear injection of foreign DNA to somatic cell nuclear transfer, intracytoplasmic sperm injection-mediated gene transfer, lentiviral transduction, and cytoplasmic injection. Mechanistically, the passive transgenesis approach based on random integration of foreign DNA was developed to active genetic engineering techniques based on the transient activity of ectopic enzymes, such as transposases, recombinases, and programmable nucleases. Whole-genome sequencing and annotation of advanced genome maps of the pig complemented these developments. The full implementation of these tools promises to immensely increase the efficiency and, in parallel, to reduce the costs for the generation of genetically engineered pigs. Today, the major application of genetically engineered pigs is found in the field of biomedical disease modeling. It is anticipated that genetically engineered pigs will increasingly be used in biomedical research, since this model shows several similarities to humans with regard to physiology, metabolism, genome organization, pathology, and aging.