Effect of amino acid substitution in New Delhi metallo-β-lactamase on carbapenem susceptibility

The aim of this work was investigation of clinically important amino acid substitutions of NDM-1 variants. A blaNDM-1 gene was cloned into expression vector pET100/D-TOPO. The sequence of NDM-1 variants with substituted amino acids was determined by ClustalW program. A pET100/D-TOPO + blaNDM-1 was used to generate the alanine mutations at different positions, such as NDM-2 (P28A), NDM-3 (D95A), NDM-4 (M154A), NDM-5 (V88A), NDM-7 (D130A), and NDM-9 (E152A). The mutant variants were transformed into Escherichia coli DH5α. Changes in the activities of alanine mutation variants were determined by E-test. All samples had 32 μg/ml MIC values against ampicillin. The 28th amino acid mutation sample had the highest MIC value against ceftazidime, whereas decreased MIC value for piperacillin. It was observed that the resistance to imipenem was increased in mutant variants D95A, M154A, D130A, and E152A, comparing with P28A and V88A. It was found that NDM-1 has 0.64 μg/ml and the 130th amino acid mutation sample has 0.75 μg/ml meropenem MIC value

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

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

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

Screening of β-lactamase resistance genes clinical isolates of Pseudomonas aeruginosa and characterization of a new metallo-β-lactamase (VIM-38)

Bu doktora tezinde, 104 P. aeruginosa suşu çalışılmıştır.Antibiyogram sonucuna göre izolatlardan 24 tanesinin çoklu ilaç-dirençli olduğu belirlendi. Tüm izolatlarda beta-laktam direnç genlerinin varlığı PZR yöntemiyle değerlendirildi ve 104 örnekten birinin GES diğerinin ise VIM pozitif olduğu tespit edildi. DNA dizi analizine göre, GES'in %100 GES-5'e, VIM tipi MBL'in ise bir aminoasit farkla (Ala265Val) %99 VIM-5'e benzediği belirlendi. Bu yeni varyant VIM-38 olarak adlandırıldı ve nükleotid sırası GenBank'a giriş yapılarak kayıt numarası alındı (Kayıt No: KC469971). blaVIM-38 pET-28a ekspresyon vektörüne klonlanarak Ni-afinite ve jel filtrasyon kromotografisi ile saflaştırıldı. Kütle spektrometresi kullanılarak VIM-5 ve VIM-38'in kütleleri belirlendi ve proteinlerin bir veya iki çinko iyonu içerdikleri doğrulandı. Aynı deneysel koşullar altında VIM-1, VIM-2 ve VIM-5 enzimleri ile karşılaştırmalı olarak VIM-38 enziminin detaylı kinetik ve biyokimyasal karakterizasyonu yapıldı. Genel olarak, VIM varyantları test edilen ß-laktam substratlara karşı katalitik etkinliklerinde çok az farklılıklar görüldü. Sefalothin ve nitrosefin tüm VIM varyantları tarafından eşit hidroliz edildi. Sefoksitin için VIM-2, VIM-5 ve VIM-38 ile VIM-1 karşılaştırıldığı zaman VIM-1 çok düşük kcat/Km değerine sahiptir ve aralarında yaklaşık 6 kat fark vardır. VIM-38 VIM-5 enzimi ile karşılaştırıldığı zaman seftazidim için yaklaşık 10 kat düşükkcat/Km değerine sahip olduğu belirlendi. VIM varyantları üzerine triptofan türevleri ile yapılan inhibisyon araştırmasında özellikle VIM-5 ve VIM-38 enzimlerinin en düşük %KA sahip olduğu görüldü. Test edilen inhibitörler tarafından VIM-1 hiç inhibe olmazken VIM-2'nin zayıf bir şekilde inhibe edildi. Enzimlerin çok yakın CD spektrasına ve sekonder yapıya sahip oldukları belirlendi. VIM-5 ve VIM-38 enzimleri VIM-1 ve VIM-2 ile karşılatırıldıkları zaman çok daha kararlı oldukları tespit edildi. In this dissertation, 104 P. aeruginosa clinical isolates were investigated. According to antibiotic resistance profile, 24 isolates were considered as a multidrug resistant. The presence of beta-lactam resistance gene in all strains evaluated using the PCR method and one of the 104 samples was determined that while the GES other VIM positive. According to DNA sequence analysis revealed that GES is the closest 100% to GES-5, VIM type MBL's is exhibiting one amino acid substitution (Ala265Val) in comparison to the closest 99%, VIM-5. This novel blaVIM variant, named VIM-38 and the nucleotide sequences of the new VIM allel was submitted to the GenBank database and can be found under accession number KC469971. blaVIM-38 cloned into the expression vector pET-28a, protein is purified by Ni-affinity and gel filtration chromatography.Mass spectrometric analysis validated the identities of VIM-5, VIM-38 and confirmed the binding of two or one metal ions for each of the proteins. Under the same experimental conditions, VIM-38 enzyme were performed with detailed kinetic and biochemical characterization of as compared to VIM-1, VIM-2 and VIM-5. Overall, the VIM variants tested showed minimal differences in their catalytic efficiencies towards the tested ß-lactam substrates. Cephalothin and nitrocefin were equally hydrolysed by all the tested VIM variants. For cefoxitin VIM-2, VIM-5 and VIM-38 compared to VIM-1 had the lowest kcat/Km values; >6 fold higher. VIM-38 had a ~10 times lowerkcat/Kmvalue for ceftazidime compared to VIM-5. The tryptophan derivatives were shown to have the lowest RA% particularly for VIM-5 and VIM-38. VIM-1 was not inhibited by any of the tested compounds while VIM-2 was only weakly inhibited. The enzymes had very similar CD spectra and secondary structures. VIM-38 and VIM-5 were the most stable variants compared to VIM-1 and to VIM-2

Antibiotic induced biofilm formation of novel multidrug resistant Acinetobacter baumannii ST2121 clone

The aim of this study was to identify antimicrobial resistance and virulence factor genes exhibited by multidrug resistant (MDR) Acinetobacter baumannii, to analyze biofilm formation and to investigate clonal subtypes of isolate. Whole genome sequencing was done by Illumina NovaSeq 6,000 platform and multilocus sequence typing (MLST) was performed by Oxford and Pasteur typing schemes. Influence of imipenem and levofloxacin on biofilm formation was investigated in 96-well plates at 3 replicates. The strain was found to carry OXA-23, OXA-51-like, AmpC and TEM-1 beta-lactamases. The sequence of the blaOXA-51-like gene has been identified as a blaOXA-66. According to Pasteur MLST scheme the strain displayed ST2 allelic profile. However, based on Oxford MLST scheme this strain represents the new ST2121, as the gdhB gene has a single allelic mutation namely, the gdhB-227. It was determined that MDR isolate carried bap, basABCDFGHIJ, csuA/BABCDE, bauABCDEF, plcD, pgaABCD, entE, barAB, ompA, abaIR, piT2EAFTE/AUBl, fimADT, cvaC, bfmR, bfmS virulence genes. In our study imipenem induced the highest biofilm formation at a concentration of 32 mg/ml and levofloxacin at a concentration of 16 mg/ml. In conclusion, we detected a new MDR A. baumannii ST2121 clone harboring blaOXA-66 gene that has been reported for the first time in Turkey

Determination of the cytotoxicity and antibiofilm potential effect of equisetum arvense silver nanoparticles

This study aimed to synthesize and characterize silver nanoparticles (AgNPs) by green synthesis from Equisetum arvense (Ea) extracts and to investigate their cytotoxicity, antibiofilm activity, and alpha-glucosidase enzyme inhibition. Diverse characterization techniques were applied to verify the production of nanoparticles. SEM examination confirmed that the size of nanoparticles is in the range of 40-60 nm. Also, interactions between silver and natural compounds of plant extract were confirmed through FT-IR and EDX analyses. It was determined that Equisetum arvense silver nanoparticles had antibiofilm activity against three different clinical strains with high biofilm-forming ability. AgNPs reduced the biofilm-forming capacity of clinical A. baumannii isolate with strong biofilm-forming capacity by approximately twofold, while the capacity of clinical K.pneumonaie and E.coli isolates decreased by 1.5 and 1.2 fold, respectively. The alpha-glucosidase enzyme inhibition potential of the AgNPs, which is determined as 93.50%, was higher than the plant extract with, and the alpha- 30.37%. MTT was performed to assess whether incubation of nanoparticles with A549 and ARPE-19 cell lines affected their viability, and a dramatic reduction in cell growth inhibition of both A549 and ARPE-19 cells was observed. It has been shown that A549 cells treated with 200 and 150 mu g/mL nanoparticles had less cell proliferation compared to control cells at 24-h and 48-h incubation time. According to these results, Ea-derived AgNPs appear to have potential anticancer activity against A549 cancer cells. Investigating the effects of green synthesis nanoparticles on microbial biofilm and various tumors may be important for developing new therapies. The outcomes of this study have showed that Ea-AgNPsmay have a high potential both in the treatment of pathogenic strains that form biofilms, as well as in anticancer therapy use

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