Evaluation of quercetin as a potential β-lactamase CTX-M-15 inhibitor via the molecular docking, dynamics simulations, and MMGBSA

Antimicrobial resistance (AMR) threatens millions of people around the world and has been declared a global risk by the World Economic Forum. One of the important AMR mechanisms in Enterobacteriaceae is the production of extended-spectrum β-lactamases. The most common ESBL, CTX-M β-lactamases, is spread to the world by CTX-M-15 and CTX-M-14. Sulbactam, clavulanic acid, and tazobactam are first-generation β-lactamase inhibitors and avibactam is a new non-β-lactam β-lactamase inhibitor. We studied that avibactam, sulbactam, clavulanic acid, tazobactam, and quercetin natural flavonoids were docked to target protein CTXM-15. Subsequently, the complexes were simulated using the molecular dynamics simulations method during 100 ns for determining the final binding positions of ligands. Clavulanic acid left CTX-M-15 and other ligands remained in the binding site after the simulation. The estimated binding energies were calculated during 100 ns simulation by the MMGBSA-MMPBSA method. The estimated free binding energies of avibactam, sulbactam, quercetin, tazobactam, and clavulanic acid were sorted as –33.61 kcal/mol, –16.04 kcal/mol, –14 kcal/mol, –12.68 kcal/mol, and –2.95 kcal/mol. As a result of both final binding positions and free binding energy calculations, Quercetin may be evaluated an alternative candidate and a more potent β-lactamases inhibitor for new antimicrobial combinations to CTX-M-15. The results obtained in silico studies are predicted to be a preliminary study for in vitro studies for quercetin and similar bioactive natural compounds. These studies are notable for the discovery of natural compounds that can be used in the treatment of infections caused by β-lactamase-producing pathogens

Next-generation sequencing of plasmid carrying blaOXA-48 in Klebsiella pneumoniae from Turkey

A carbapenem-resistant Klebsiella pneumoniae strain was isolated in Turkey in 2012 and blaNDM-1 and blaOXA-48 genes were observed in this strain. The aim of this study was to investigate transferability of plasmid bearing blaOXA-48 in K. pneumoniae and to use whole-genome sequencing in order to understand the genetic context of plasmid. K. pneumoniae strain was used as donor in conjugation experiments. Antibiotic susceptibility profile of selected transconjugant was determined. Plasmid was isolated from transconjugant colony and was named as pKPT. Complete sequencing of the pKPT was conducted using a next-generation sequencing. Annotation of the contigs was performed using the Geneious R9, followed by finding open reading frames (ORFs) with selected web-based tools. BLAST analysis was performed at the NCBI BLAST server to determine genes showing more than 90% similarity with these ORFs. Results of antibiotic susceptibility test showed that transconjugant colony was resistant to ampicillin/ sulbactam, piperacillin, and piperacillin/tazobactam. The pKPT plasmid had a length of 45,217 bp and an average G + C content of 49%. Blast analysis revealed that pKPT was included in the IncL/M incompatibility group. The pKPT was found to contain blaOXA-48 within Tn1999.2 transposon without any other antibiotic resistance gene.Gumushane University: BAP-17 F5119.02.0

Antimicrobial resistance patterns and integron carriage of Escherichia coli isolates causing community-acquired infections in Turkey

We aimed to observe antimiCrobial resistance patterns and integron carriage of Escherichia coli isolates causing community-acquired infections. Two hundred sixty-eight E. coli strains were obtained from outpatients with various infections at different polyclinics at the 82nd Year of State Hospital in Rize, Turkey. Susceptibility to antimicrobials was tested using a disk diffusion method. The presence of integrons was examined using PCR with specific primers. Positive PCR results were confirmed by sequencing. A broth mating method was used for conjugation assays. Extragenic palindromic-FOR was performed using the oligonucleotide primer BOXA1R. Resistance frequency for ampicillin,trimethoprim/sulfamethoxazole, and tetracycline was determined as 50.6%, 33.5%, and 36.8% respectively. No strains were resistant to amikacin. Seventy isolates were positive for the intll gene, of which 49 carried gene cassettes. Eleven isolates were positive for the int12 gene, eight of swhich carried gene cassettes. Seven gene cassettes (dfrAl, dfrA5, dfrA7, dfrA17, aadAl, aadA5, and sat2) were predominantly harbored in integrons. We detected conjugative plasmids harboring integrons in two E. coli strains. Four strain clusters were yielded by BOX-FOR fingerprints showing that they were clonally related. No apparent relationship occurred among class 1 and 2 integron-carrying strains. We conclude that integrons are widespread in genetically variable E. coli strains and will continue to mediate dissemination of resistance genes in the community.We aimed to observe antimiCrobial resistance patterns and integron carriage of Escherichia coli isolates causing community-acquired infections. Two hundred sixty-eight E. coli strains were obtained from outpatients with various infections at different polyclinics at the 82nd Year of State Hospital in Rize, Turkey. Susceptibility to antimicrobials was tested using a disk diffusion method. The presence of integrons was examined using PCR with specific primers. Positive PCR results were confirmed by sequencing. A broth mating method was used for conjugation assays. Extragenic palindromic-FOR was performed using the oligonucleotide primer BOXA1R. Resistance frequency for ampicillin, trimethoprim/sulfamethoxazole, and tetracycline was determined as 50.6%, 33.5%, and 36.8% respectively. No strains were resistant to amikacin. Seventy isolates were positive for the intll gene, of which 49 carried gene cassettes. Eleven isolates were positive for the int12 gene, eight of swhich carried gene cassettes. Seven gene cassettes (dfrAl, dfrA5, dfrA7, dfrA17, aadAl, aadA5, and sat2) were predominantly harbored in integrons. We detected conjugative plasmids harboring integrons in two E. coli strains. Four strain clusters were yielded by BOX-FOR fingerprints showing that they were clonally related. No apparent relationship occurred among class 1 and 2 integron-carrying strains. We conclude that integrons are widespread in genetically variable E. coli strains and will continue to mediate dissemination of resistance genes in the community.This work was supported by Recep Tayyip Erdogan University Research Fund grants BAP-2012.106.01.11 and BAP-2011. 102.03.3

Cloning, expression, and characterization of a novel CTP synthase gene from Anoxybacillus gonensis G2

The cytidine-5'-triphosphate (CTP) synthase (EC 6.4.3.2) gene (pyrG) was cloned and sequenced from the thermophilic bacterium Anoxybacillus gonensis G2 (Ago). The gene is 1590 bp in length and encodes a protein of 530 amino acids, with a molecular mass of 59.5 kDa. The amino acid sequence of CTP synthase shares approximately 90%–94% similarity to Bacillus sp., and it belongs to the triad glutamine amidotransferases, which utilize a Cys–His–Glu triad for activity. Multiple sequence alignments revealed that the enzyme includes conserved amino acids responsible for catalytic activity and the binding of a divalent metal ion (Mg+2). AgoCTP synthase (AgoG2CTPs) was overproduced in Escherichia coli BL21 (DE3) pLysS as recombinant and purified by nickel affinity chromatography. Its biochemical characterization showed that the enzyme had maximal activity at pH 9.0–10.0 and 65 ºC. Km, Vmax, and kcat were found to be approximately 12.415 mM, 0.381 U/L, and 0.762 s–1 at 65 ºC, respectively. CTP synthase promotes the formation of CTP in dividing cells and is a recognized target for anticancer and antibacterial drugs. The results obtained from this study can be improved upon with the use of different species and substrates

Investigation of class 1 integrons with antibiotic resistance genes in multidrug-resistant acinetobacter baumannii strains and determination of plant extract effects on multidrug-resistant isolates

This study aimed to investigate the presence of resistance genes in multidrug-resistant A. baumannii isolates as well as to determine the antibacterial activity of selected plant extracts against isolates. 41 strains were isolated from various clinical samples. PCR tests were performed using the primers. Methanol was used as solvent for the preparation of the plant extracts. MIC values of the plant extracts were determined by the broth microdilution method. The bla(OXA-)(23), bla(CTX-M-1), bla(CTX-M-2), bla(GES) genes and Class 1 integrons were detected in five isolated strains. The lowest MIC value (2.25 mg/mL) was determined for the Echinacea putpurea extract, while the highest MIC value (50 mg/mL) was determined for the Morus alba extract. Determination of the antibacterial effect of plants extracts used in the study against A. baumannii isolates shows the importance of screening the antibacterial activity of plants in the fight against antibiotic resistance.Studiul a avut ca scop investigarea genelor de rezistență ale izolatelor de A. baumannii multirezistente, precum și determinarea activității antibacteriene a unor extracte vegetale supra a 41 specii microbiene izolate din diferite probe clinice. Metanolul a fost folosit ca solvent de extracție. Valorile concentrației minime inhibitorii ale extractelor obținute au fost determinate prin metoda microdiluției. Genele blaOXA-23, blaCTX-M-1 , blaCTX-M2 și blaGES, alături de integroni din clasa 1 au fost decelate pentru 5 specii izolate. Valoarea MIC cea mai scăzută (2,25 mg/mL) a fost obținută pentru extractul din Echinaceea purpurea, în timp ce cea mai mare valoare MIC (50 mg/mL) a fost determinată pentru extractul de Morus alba. Determinarea efectului antibacterian ale extractelor vegetale împotriva izolatelor de A. baumannii arată importanța screeningului activității antibacteriene a plantelor în lupta împotriva rezistenței la antibiotice

Isolation, identification and characterization of biotechnologically important bacteria from microflora of Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae)

The chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) is one of the most important insect pests of chestnut. The aim of this study was to isolate and characterize bacteria from D. kuriphilus to obtain new microbial agents for both biological control and other biotechnological applications. D. kuriphilus larvae were collected from chestnut fields located in Bursa and Yalova provinces of Marmara Region of Turkey during May-July 2014. Four bacterial isolates were obtained from D. kuriphilus. According to their morphological, biochemical and molecular properties, these isolates were identified as Staphylococcus saprophyticus (Dk1), Paenibacillus sp. (Dk2), Pseudomonas flourescens (Dk3) and Paenibacillus sp. (Dk4). To the best of our knowledge, this is the first study on the bacterial flora of D. kuriphilus. In our study, the potential of these isolates as a biological control agent against different hazardous pests and other possible biotechnological applications of importance were discussed under the light of literature

OXA- and GES-type beta-lactamases predominate in extensively drug-resistant Acinetobacter baumannii isolates from a Turkish University Hospital

This study was supported by grants from Recep Tayyip Erdogan University (BAP-2012.106.01.11 and BAP-2011.102.03.3). AYP was supported by the Australian National Health and Medical Research Council (APP1047916 and APP1010114).We determined the antibiotic susceptibility and genetic mechanisms of resistance in clinical strains of Acinetobacter baumannii from Istanbul, Turkey. A total of 101 clinical strains were collected between November 2011 and July 2012. Antimicrobial susceptibility was performed using the Vitek 2 Compact system and E-test. Multiplex PCR was used for detecting bla(OXA-51-like), bla(OXA-23-like), bla(OXA-40-like) and bla(OXA-58-like) genes. ISAba1, bla(IMP-like), bla(VIM-like), bla(GES), bla(VEB), bla(PER-2), aac-3-Ia and aac-6'-Ib and NDM-1 genes were detected by PCR and sequencing. By multiplex PCR, all strains were positive for bla(OXA-51), 79 strains carried bla(OXA-23) and one strain carried bla(OXA-40). bla(OXA-51) and bla(OXA-23) were found together in 79 strains. ISAba1 element was detected in 81 strains, and in all cases it was found upstream of bla(OXA-51). GES-type carbapenemases were found in 24 strains (GES-11 in 16 strains and GES-22 in 8 strains) while bla(PER-2), bla(VEB-1), bla(NDM-1), bla(IMP)- and bla(VIM)-type carbapenemases were not observed. Aminoglycoside modifying enzyme (aac-3-Ia and aac-6'Ib) genes were detected in 13 and 15 strains, respectively. Ninety-seven (96%) A. baumannii strains were defined as MDR and of these, 98% were extensively drug resistant (sensitive only to colistin). Colistin remains the only active compound against all clinical strains. As seen in other regions, OXA-type carbapenemases, with or without an upstream ISAba1, predominate but GES-type carbapenemases also appear to have a significant presence. REP-PCR analysis was performed for molecular typing and all strains were collected into 12 different groups. To our knowledge, this is the first report of GES-11 and OXA-40 in A. baumannii from Turkey

Chronic restraint stress impairs spatial memory while decreasing hippocampal BDNF levels in rats

Aim: This study investigated the potential role of chronic restraint stress (CRS) on spatial memory, recognition memory, brain-derived neurotrophic factor (BDNF) and acetylcholine (ACh) levels in young adult rats. Material and Methods: In the study, 16 female rats of 12 weeks old were used. Rats were divided into two groups as control and CRS (n=8). CRS was applied 5 hours a day for 21 days. Following the end of CRS, recognition memory of rats was evaluated with new object recognition test (NORT) and spatial memory was evaluated with Morris water maze (MWM) test. At the end of the study, rats were euthanized and hippocampal tissue homogenates were obtained. Hippocampal BDNF and ACh levels were determined by ELISA method. Results: Exposure to CRS did not significantly change the exploratory behavior and discrimination index of rats (p > 0.05). In the test phase in which spatial memory was evaluated, CRS decreased the time spent in the target quadrant (p > 0.01). There was no significant difference between days in the training phase. CRS significantly decreased BDNF level in hippocampus (p > 0.05). Hippocampal ACh levels were not statistically significant (p > 0.05). Conclusions: CRS weakened cognitive functions in rats. This effect was mainly accompanied by a decrease in hippocampal BDNF levels. Our findings point to the potential role of BDNF in understanding the molecular mechanism of CRS-induced cognitive impairment

The first study on bacterial flora and biological control agent of the little spruce sawfly, Pristiphora abietina (Christ.) (Hymenoptera: Tenthredinidae)

The aim of this study was to determine the bacterial flora of Pristiphora abietina and to find the performance of the members of this flora as a biocontrol agent for this pest. For this purpose, eleven bacteria were isolated from living, diseased and dead larvae. Morphological and biochemical properties, metabolic enzyme profiles by BIOLOG microtiter plate system and total cellular fatty acid profile by Microbial Identification Systems (MIS) of the bacterial isolates were determined. In addition, 16S rRNA gene sequence analysis was performed. The isolates were identified as Bacillus pumilus (Pa1), Lysinibacillus fusiformis (Pa2, Pa10), Stenotrophomonas maltophilia (Pa3), Acinetobacter johnsonii (Pa4, Pa9), Bacillus cereus (Pa5), Rhodococcus sp. (Pa6), Staphylococcus sciuri (Pa7), Ralstonia pickettii (Pa8), Neisseria perflava (Pa11). All these bacteria were tested against P. abietina larvae. The highest insecticidal activity was obtained from S. maltophilia and L. fusiformis (65.47%, 60.71%, respectively), (p < 0.05), whereas the lowest insecticidal activity (17.26%) was obtained from N. perflava within seven days. Our result indicates that L. fusiformis (Pa2, Pa10) show potential to be used as biological control agents of P. abietina.Artvin Coruh University: ACU-BAP: 2011.F15.02.1

Agomelatin yetişkin sıçanlarda skopolamin kaynaklı öğrenme ve hafıza bozukluğunu tersine çevirir

Amaç: Agomelatin, melatonin reseptör (MT1 ve MT2) agonisti ve serotonin reseptör (5-HT2C) antagonisti olan antidepresan bir ilaçtır. Artan kanıtlar, agomelatinin nöroprotektif ve nöromodülatör etkiye sahip olduğunu göstermektedir. Bu çalışmada skopolamin indüklü bilişsel yetmezlik oluşturulan sıçanlarda agomelatinin potansiyel etkileri araştırılmıştır. Materyal ve Metot: Erişkin erkek sıçanlara 21 gün süreyle skopolamin (1 mg/kg) ve agomelatin (40 mg/kg) uygulandı. İlaç uygulamalarını takiben sıçanlar bilişsel davranışların değerlendirilebilmesi amacıyla yeni nesne tanıma (YNT) ve Morris su labirenti (MSL) testine tabi tutuldu. İlave olarak, beyin nörokimyasal analizleri için hipokampus ve prefrontal kortekste beyin-türevi nörotrofik faktör (BDNF) ve asetilkolin (ACh) düzeyleri değerlendirildi. Bulgular: Skopolamin hem uzamsal hafızayı hem de ayırt etme indeksini önemli ölçüde azalttı (p<0,05). Agomelatin tedavisi uzamsal hafıza performansını ve keşif süresini arttırdı, ancak ayrımcılık indeksini etkilemedi (P>0,05). Ayrıca agomelatin, skopolamin grubuna kıyasla hem hipokampusta hem de prefrontal kortekste BDNF düzeylerini önemli ölçüde arttırdı (sırasıyla p<0,05, p<0,01). Diğer yandan grupların ACh düzeyleri arasında istatistiksel olarak anlamlılık bulunmadı (p>0,05). Sonuç: Birlikte ele alındığında, bu sonuçlar agomelatinin skopolamin kaynaklı hafıza yetmezliğinin hafifletilmesinde belirgin rol oynadığını göstermiştir. Bu nedenle, agomelatinin bilişsel yetmezliğin önlenmesinde potansiyel bir ajan olabileceğini öne sürüyoruzObjective: The antidepressant agomelatine agent is a melatonin receptor (MT1 and MT2) agonist and a serotonin receptor (5-HT2C) antagonist. Increasing evidence shows that agomelatine has neuroprotective and neuromodulatory effects. In this study, the potential effects of agomelatine in rats with scopolamine-induced cognitive impairment were investigated. Materials and Methods: Adult male rats were administered scopolamine (1 mg/kg) and agomelatine (40 mg/kg) for 21 days. After drug administration, rats were subjected to new object recognition (NOR) and Morris water maze (MWM) tests in order to evaluate cognitive behaviors. In addition, brain-derived neurotrophic factor (BDNF) and acetylcholine (ACh) levels in the hippocampus and prefrontal cortex were evaluated. Results: Scopolamine significantly decreased both spatial memory and discrimination index (p<0.05). Agomelatine treatment increased spatial memory performance and exploration time, but did not affect the discrimination index (P>0.05). In addition, agomelatine significantly increased BDNF levels in both hippocampus and prefrontal cortex compared to the scopolamine group (p<0.05, p<0.01, respectively). On the other hand, there was no statistically significant difference between the ACh levels of the groups (p>0.05). Conclusion: Taken together, these results demonstrated that agomelatine plays a important role in alleviating scopolamine-induced memory impairment. Therefore, we suggest that agomelatine may be a potential agent in the prevention of cognitive impairment