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

Antibiotic resistance profiles of enteric cacteria isolated from Kucukcekmece Lagoon (Istanbul-Turkey)

Sivri, Nuket/0000-0002-4269-5950; SANDALLI, Cemal/0000-0002-1298-3687WOS: 000314625500019The aim of this study is to find out the density of the fecal bacteria and to analyze resistance to antimicrobials of Gram negative bacilli isolated from the Kucukcekmece Lagoon, Istanbul. Samples were taken monthly from June 2006 to June 2008 and a total of 232 Gram negative bacilli were isolated. Chloramphenicol, tetracycline, nalidixic acid, ampicillin, imipenem, ceftazidime, amikacin, streptomycin and amoxicillin + clavulanic acid were used in antimicrobial susceptibility tests. Susceptibility to trimethoprim + sulfamethoxazole was also examined in only integron-bearing organisms. the antibiotic resistance tests resulted in bacteria being the most resistant against ampicillin (76.29%) and the most sensitive against amikacin (93.56%). of 232 isolates, 20 (8.6%) coliforms harbored class 1 and/or class 2 integrons. DNA sequencing showed that variable regions of the integrons harbored various gene cassettes; dfrA12, dfrA15, dfrA17, aadA1, aadA2, aadA5, b/a(OXA-30) and sat2. Integrons were found in bacteria from all sampling areas except 12 and D3. in this study, the determination of bacterial identification of the species of Gram negative bacilli and their Antibiotic Resistance Profiles in the Kucukcekmece Lagoon for the first time was investigated. A finding indicates that there is a heavy fecal pollution in this lagoon environment, which might probably be resulted from the intensive anthropogenic facilities. the risk to public health could be the transfer of antibiotic resistance determinants from the bacterial isolates to normal microbiota bacteria of humans unless the effective precautions such as water treatment plants are taken.Research Fund of the University of IstanbulIstanbul University [BAP-543/05052006]; TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [105Y116]This work was supported by the Research Fund of the University of Istanbul (Project Number : BAP-543/05052006) and the TUBITAK (Project Number : 105Y116)

Evaluation of genetic diversity of cultivated tea clones (Camellia sinensis (L.) Kuntze) in the eastern black sea coast by inter-simple sequence repeats (ISSRS)

SANDALLI, Cemal/0000-0002-1298-3687; PEHLIVAN, NECLA/0000-0002-2045-8380WOS: 000376744800008Tea is the most globally consumed drink after spring water and an important breeding plant with high economical value in Turkey. in half a century, various kinds of tea cultivars have been bred in Turkey to improve the quality and yield of tea plants. Since tea reproduces sexually, tea fields vary in quality. Thus, determining the genetic diversity and relationship of the plants to support breeding and cultivation is important. in this study we aimed to determine the genetic diversity of tea cultivars breeding in the Eastern Black Sea coast of Turkey and the genetic relationship between them, to verify whether the qualitative morphological designations of the clones are genetically true by the ISSR markers. Herein, the genetic diversity and relationships of 18 Turkish tea cultivars were determined using 15 ISSR markers with sizes ranging from 250 to 3000 base pairs. the similarity indices among these cultivars were between 0.456 and 0.743. Based on cluster analysis using UPGMA, some of tea cultivars originating from the same geographical position were found to be clustered closely. Our data provide valuable information and a useful basis to assist selection and cloning experiments of tea cultivars and also help farmers to find elite parental clones for tea breeding in the Eastern Black Sea coast of Turkey.Recep Tayyip Erdogan UniversityRecep Tayyip Erdogan University [BAP_2013.102.03.4]This work was supported by Recep Tayyip Erdogan University Research Fund Grants BAP_2013.102.03.4

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

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

Comparison of verona integron-borne metallo-beta-lactamase (VIM) variants reveals differences in stability and inhibition profiles

DUZGUN, AZER OZAD/0000-0002-6301-611X; Abboud, Martine I./0000-0003-2141-5988; Brem, Jurgen/0000-0002-0137-3226; McDonough, Michael A/0000-0003-4664-6942; Rydzik, Anna/0000-0003-3158-0493; DUZGUN, AZER OZAD/0000-0002-6301-611X; McDonough, Michael/0000-0003-4664-6942; Schofield, Christopher/0000-0002-0290-6565; SANDALLI, Cemal/0000-0002-1298-3687WOS: 000376490800025PubMed: 26666919Metallo-beta-lactamases (MBLs) are of increasing clinical significance; the development of clinically useful MBL inhibitors is challenged by the rapid evolution of variant MBLs. the Verona integron-borne metallo-beta-lactamase (VIM) enzymes are among the most widely distributed MBLs, with > 40 VIM variants having been reported. We report on the crystallographic analysis of VIM-5 and comparison of biochemical and biophysical properties of VIM-1, VIM-2, VIM-4, VIM-5, and VIM-38. Recombinant VIM variants were produced and purified, and their secondary structure and thermal stabilities were investigated by circular dichroism analyses. Steady-state kinetic analyses with a representative panel of beta-lactam substrates were carried out to compare the catalytic efficiencies of the VIM variants. Furthermore, a set of metalloenzyme inhibitors were screened to compare their effects on the different VIM variants. the results reveal only small variations in the kinetic parameters of the VIM variants but substantial differences in their thermal stabilities and inhibition profiles. Overall, these results support the proposal that protein stability may be a factor in MBL evolution and highlight the importance of screening MBL variants during inhibitor development programs.Rhodes Trust; Scientific and Technology Council of Turkey; Recep Tayyip Erdogan Universitesi Research FundRecep Tayyip Erdogan University [BAP-2013.102.03.13]; Medical Research CouncilMedical Research Council UK (MRC) [MR/L007665/1]; Medical Research Council/Canadian Grant [G1100135]; Biochemical Society Krebs Memorial Award; Medical Research CouncilMedical Research Council UK (MRC) [G1100135, MR/N002679/1] Funding Source: researchfishThe Rhodes Trust provided funding to Anne Makena. Scientific and Technology Council of Turkey provided funding to Cemal Sandalli. Recep Tayyip Erdogan Universitesi Research Fund provided funding to Aysegul Saral, Aysegul C. Cicek, and Cemal Sandalli under grant number BAP-2013.102.03.13. Medical Research Council provided funding to Jurgen Brem, Michael A. McDonough, Anna M. Rydzik, and Christopher J. Schofield under grant number MR/L007665/1. Medical Research Council/Canadian Grant provided funding to Jurgen Brem, Michael A. McDonough, Anna M. Rydzik, and Christopher J. Schofield under grant number G1100135. Biochemical Society Krebs Memorial Award provided funding to Martine I. Abboud

Characterızatıon of catalytıc carboxylate trıad ın non-replıcatıve dna polymerase III (pol E) of Geobacillus kaustophilus HTA

SANDALLI, Cemal/0000-0002-1298-3687WOS: 000327446000008PubMed: 23273190Three aspartic acid residues D378, D380 and D531 form the catalytic carboxylate triad in Geobacillus kaustophilus (Gka) DNA polymerase III alpha-subunit homolog, pol E. We cloned, expressed and purified wild type (WT), alanine (D -> A) and glutamate (D -> E) mutant enzymes of D378, D380 and D531. the WT and mutant enzymes were biochemically characterized for DNA binding, dNTP binding and catalytic activity in the presence of two metal ions (Mg2+ and Mn2+). the polymerase activity of all mutant enzymes was lost in the presence Mg2+, whereas D378E and D531E mutant enzymes showed about 35 and 60 percent activity, with Mn2+. D380E mutant enzyme did not show noticeable activity with either metal ions suggesting its absolute requirement in polymerase reaction. Kinetic characterization of individual mutant proteins showed that the template-primer binding affinity (K-D.DNA) did not change due to both D -> A or D -> E mutation. the K-M.dNTP for D378E and D531E increased by about 10- and 100-fold, compared to WT enzyme implicating the function of these residues in dNTP binding. Based on these results and the analysis of the available crystal structures of the homologous enzyme species in their apo and E. DNA. dNTP ternary complex forms, we conclude that D378 and D531 are mainly responsible for the binding of metal chelated substrate dNTP, while D380 is solely responsible for the chemical step of phosphodiester bond formation.TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK)This study was supported by a scholarship to C. Sandalli from TUBITAK

Characteristics in the whole-genome sequence of Klebsiella pneumoniae ST147 from Turkey

The study aimed to analyze antibiotic resistance determinants in a carbapenem-resistant Klebsiella pneumoniae by whole-genome sequencing (WGS). K. pneumoniae was isolated from a urine sample and it was characterized by 16S rDNA sequencing in Turkey. This strain was named as Kpn Rize-53-TR. Antimicrobial susceptibility testing was performed for seventeen antibiotics by VITEK-2 and the result was confirmed by MIC. The whole genome of isolate was sequenced by lllumina and was analysed by bioinformatic tools for MIST, replicon types, and antimicrobial resistance genes. The whole genome data was submitted to NCBI. The isolate was found to be resistant to all tested beta-lactam antibiotics and the highest MIC values were found for piperacillin, piperacillin/tazobactam (>= 128). No resistance to colistin and moderate susceptibility to amikacin and tetracycline was observed. The isolate carried 12 resistance genes belonging to 10 resistance classes; ere(A), fosA, oqxtl, cmlA1, aac(a)-IIa, bla(KPC-2), bla(TEM-)(1A), bla(SHV)(-)(67), bla(CTX-M-)(15), bla(OX)(A-1-2-9). Mutations were detected in gyrA (83Y) and parC (80I) genes. Clonal subtype of the isolate was ST147, and it had wzi420 and wzc38 alleles. Its serotype was O3/O3a. The bla(KPC-2) was firstly found in both ST147 clonal group in Turkey and in serotype O3/O3a in the world. By plasmid replicon typing, five plasmids IncEII(K), Col(BS512), IncR, IncFIA(HI1) and IncFIB(pQil) were determined in Kpn Rize-53-TR and bla(KPC)(-2) was located on IncFII(K) plasmid. The presence of bla(K)(PC)(-2) on the plasmid with other resistance genes accelerates its own spread together with other resistance genes

M-MLV reverse transcriptase was strongly inhibited by essential oil extract of paeonia daurica subsp. macrophylla

Paconia species arc known for their rich chemical content and their compounds exhibit different medicinal properties. In this study, we firtly investigated the essential oil (EO) content of the root, scape, fruit and fruit bark of Paeonia daurica Andrews subsp, Macrophylla Albow (P, daurica) by using gas chromatography-mass spectrometry (GC-MS). Both solid-phase micro-extraction (SPME) and hydro-distillation (HD) were used to prepare EO extracts for each plant sample. While fifty-two compounds were identified in SPME-EO extract, sixty-eight compounds were identified in HD-EO extract and eighteen compounds were jointly found in both extract. Major constituents of the EOs were salicylaldehyde (100-65%), myrtanal (3.10-45%), palmitic acid (2.1%-40.4%), methyl salicylate (3.1%-37.5%) and myrtenal (3%-35.1%). Fruit bark had the richest identified EOs among plant parts. Furthermore, the total SPME-EO and HD-EO extracts were evaluated for their inhibition on Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV-RT) by using primer extension assay (measuring of single dATP nucleotide addition). M-MLV-RT could not add a single nucleotide to the 14-mer primer in the presence of both EO extracts and it revealed that both SPME and HD total EO extracts had strong inhibition effect on M-MLV-RT. alpha-bisabolol, beta-bisabolol and benzoic acid-ethylester (a derivate of benzoic acid) in HD-fruit extract were evaluated as potential compounds responsible for the inhibitory effect according to literature, To the best of our knowledge, this is the first detailed report about EO compositions and its inhibition effect against M-MLV-RT of the root. scape. fruit and fruit bark of P. daurica. We propose the EOs of P. daurica might have potential of therapeutic candidate for preventing viral diseases.Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) TUBITAK-113Z05

Determination of a novel integron-located variant (bla(OXA-320)) of Class D beta-lactamase in Proteus mirabilis

This work was partially supported by Recep Tayyip Erdogan University Research Fund Grants BAP-2009.102.03.2 and BAP-2011.102.03.3.Proteus mirabilis (P. mirabilis) is one of Gram-negative pathogens encountered in clinical specimens. A clinical isolate (TRP41) of P. mirabilis was isolated from a Turkish patient in Turkey. The isolate was identified using the API 32GN system and 16S rRNA gene sequencing and it was found resistant to ampicillin/sulbactam, piperacillin, tetracycline, and trimethoprim/sulfamethoxazole. This isolate was harboring a Class 1 integron gene cassette and its DNA sequence analysis revealed a novel bla(OXA) variant exhibiting one amino acid substitution (Asn266Ile) from bla(OXA-1). This new variant of OXA was located on Class 1 integron together with aadA1 gene encoding aminoglycoside-modifying enzymes. According to sequence records, the new variant was named as bla(OXA-320). Cassette array and size of integron were found as bla(OXA-320)-aadA1 and 2086bp, respectively. The bla(OXA-320) gene is not transferable according to conjugation experiment. In this study, we report the first identification of bla(OXA-320)-aadA1 gene cassette, a novel variant of Class D -lactamase, in P. mirabilis from Turkey