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

The evaluation of chromogenic medium in identification of candida species isolated from various clinical samples

Amaç: Bu çalışmada çeşitli klinik örneklerden izole edilen mayaların tür düzeyinde tanımlanmasında konvansiyonel mikolojik yöntemler ile Kromojenik besiyeri (Oxoid Brill iance™ Candida agar, İngiltere) (KB) kullanılarak yapılan tanımlamaların karşılaştırılması am açlandı. Yöntem : Otuz üç idrar, 34 transtrakeal aspirat (TTA), 7 bro n koalvaolar lavaj (BAL) örneği, 13 balgam, 12 kan, 9 yara, üç kateter ve iki vajinal akıntı olmak üzere toplam 113 klinik örnek çalışmaya alındı. İzole edilen suşların konvansiyonel mikolo jik yöntemlerle; mikroskopik ve makroskopik mo rfoloji, germ tüp testi, üre hidrolizi, ka rbonhidrat kullanımı (API 20 C AUX, Biomerieux, Fransa), üreme ısısı ve sikl o heksimid hasasiyetine göre tür tanı mlaması yapıldı. Suşlar kromojenik besiyerine (Oxoid Bri ll iance™ Candida agar, İngiltere) ekilerek renk değişimleri değerlendirildi ve her iki yöntem karşılaştırıldı. Bulgular: Çalışılan klinik örneklerde her iki yöntemle to plam 113 örneğin 103’ünde (% 91.2) aynı tür tanımlaması yapılırken,10 (%8.8) tanesinde farklı tür tanımlanmıştır. Kromejenik besiyeri C. albicans suşlarını %94.1, C. paraps ilosis %88.9 , C. glabrata %81.3 oranında doğru tanımlarken birer suş olan C. krusei ve C. tropicalis doğru, C. lusitania yanlış tanımlanmıştır. Stok kültürlerimizin 6 tan esinden birinin bakteri ile 5’inin iki çeşit Candida türü ile karışık kültür olduğu kromojenik besiyeri ile anlaşılmıştır. Sonuç : İnfeksiyon etkeni olarak sık karşılaşılan Candida suşlarının tür düzeyinde tanımlanmasında ve karışık kü ltürlerin ayırt edilme sinde kromojenik besiyerinin duyarlıl ığının yüksek olmasına rağmen nadir karşılaşılan maya türl e ri için daha fazla çalışmalara gereksinim vardır.Objective : To compare of conventional mycologic methods used to determine yeast or candida spp extracted from some clinical samples with the definitions identified using the chromogenic medium (CM, Oxoid Brilliance™ Candida agar, United Kingdom) was aimed in this study. Method : Total of 113 clinical samples (33 of urine, 34 of transtracheal aspirate (TAT), 7 of bronchoalveolar lavage (BAL), 13 of sputum, 12 of blood, 9 of wound, 3 of catheter, 2 of vaginal smear) were included. Species identification was carried out using convantional mycologic methods; microscopic and macroscopic morphology, germ tube test, urea hydrolysis, carbonhydrate use (API 20C AUX, Biom erieux, France), growth temperature and cycloheximide sensitivity. The strains were inoculated in the chromogenic medium (Oxoid Brilliance™ Candida agar, UK) and colour changes were evaluated, and two method s were comp ared. Results : With both method, 91.2% of (n=103) total 113 samples was identified as similar whilest 8.8% of them (n=10) was defined differently. 94.1% of Candida albicans spp, 88.9% of C. parapsilosis and 81.3% of C. glabrata was truly identi fied. C. krusei and C. tropicalis were correctly identified whereas C. lusitania was wrongly identified. It was understood that one of total 6 of our stock cultures was found as mixed culture with bacteria, and the rest of them was found as mixed with two types of Candida sp ecies, with using chromogenic medium. Conclusion : There is a need to further studies for rare yeast species although the sensitivity of chromogenic medium is high for using the determination of common candida sp ecies as infectious agents.

Klinik Örneklerden izole Edilen Acinetobacter baumannii ve Pseudomonas aeruginosa Suşlarinin Sinif 1 ve 2 integran Taşiyiciligi ve Yeni Bir Gen Kaseti Birlikteliği: Bla0XA-11-cmlA7

PubMed: 24506715The dissemination of antibiotic resistance genes between bacteria leads to serious problems in the treatment of infectious diseases. It has been shown that resistance genes can also be carried by the integrons. There are limited studies regarding the carriage of class 1 and 2 integrons in Adnetobacter baumannii and Pseudomonas aeruginosa clinical strains in Turkey. The aims of this study were to investigate the carriage rates of class 1 and class 2 integrans in A.baumannii and P.aeruginosa strains isolated from clinical samples in Abant Izzet Baysal University Hospital, and to characterize the antibiotic resistance gene cassettes in these integrans by sequence analyses. A total of 137 strains (77 Abaumannii and 60 P.aeruginosa) isolated from various clinical specimens (56% were sputum, 19% wound, 11 % urine, 11 % blood, 3% catheter), between March 2010-December 2012, were included in the study. The identification and antibiotic susceptibility tests of the isolates were performed by Vitek 2 Compact (bioMérieux, France) and BD Phoenix 100 (Becton Dickinson, USA) systems. The presence of intégrons were screened by PCR method using specific primer pairs targeting class 1 (intll) and 2 (intl2) integrase regions. All the samples that revealed integran amplification were subjected to DNA sequence analysis, both in the forms of cloned products and PCR amplicons. In the study, the highest susceptibility rates were found against colistin 96% and tigecycline 78% in A.baumannii, and against piperacillin/tazobactam 97% and piperacillin 93% in P.aeruginosa isolates. The highest resistance rate was determined for piperacillin/tazobactam 95% in A.baumannii strains. The presence of intll gene was detected in 33% (26/77) of A.baumannii and 10% (6/60) of P.aeruginosa isolates. When variable regions in intl1 positive strains were amplified by PCR, eight (8/77, 10%) A.baumannii and three (3/60, 5%) P.aeruginosa strains were found to harbor antibiotic resistance gene cassettes. Intl2 gene was not detected in any of the isolates. Resistance to piperacillin/tazobactam, ceftazidime, cefepime, ceftriaxone and ampicillin/sulbactam was detected as the common resistance pattern in all integron-positive A.baumannii strains, whereas resistance to ceftazidime, gentamicin and ciprofloxacin was the common pattern in all integron-positive P.aeruginosa strains. DNA sequence analysis of variable regions of integrons indicated that two separate gene cassette arrays (aacCl-aadA1 and aac(3)-1) were carried by A.baumannii strains, and two types of gene cassette arrays (bla QXA-30-aadA1 and bla0XA-11- cmlA7) were carried by P.aeruginosa strains. To our best knowledge, this is the first report of the gene sequence of blaQXA-11-cmlA7 defined in an integron gene cassette of P.aeruginosa

Characterization of class 1 and class 2 integron gene cassettes in Escherichia coli strains isolated from urine cultures: A multicenter study [İdrar Kültürlerinden İzole Edilen Escherichia coli Suşlarinda Sinif 1 ve Sinif 2 İntegron Gen Kasetlerinin Karakterizasyonu: Çok Merkezli Bir Çalişma]

PubMed ID: 27175490Escherichia coli is the most common pathogen isolated from both nosocomial and community acquired urinary tract infections. Although there are many studies from different centers concerning the antibiotic susceptibility of E.coli isolates in Turkey, the studies are quite few about class 1 and class 2 integron cassettes in clinical E.coli isolates from urinary samples. The aim of the study was to investigate the antibiotic susceptibility and the carriage of integron gene cassettes in E.coli strains isolated from urinary samples. A total of 626 E.coli strains isolated from urine cultures in microbiology laboratories located at 10 provinces from different regions of Turkey (Denizli, Ankara, Kayseri, Nigde, Şanliurfa, Kahramanmaras, Tokat, Malatya, Konya and Trabzon) between June 2011-June 2012 were included in the study. The identification and antibiotic susceptibility testing of the isolates were studied by conventional methods as well as Vitek® 2 Compact (bioMérieux, France) and BD Phoenix™ 100 (Becton Dickinson, USA) systems. The antibiotic susceptibilities of all the isolates were retested by Kirby-Bauer disk diffusion method according to CLSI recommendations in the main center of the study in order to achive the standardization. The presence of integrons was detected with polymerase chain reaction (PCR) method by using specific primers targeting class 1 (intl 1) and class 2 (intl 2) integrase gene regions. After integron amplification the samples were cloned and subjected to DNA sequencing. When the antibiotic susceptibility of the isolates were evaluated, the highest resistance was observed against most commonly used empirical antibiotics namely ampicillin and trimethoprim-sulfamethoxazole (SXT) with the mean rate of 58.6% (range: 43.8%-73.2%) and 41.2% (range: 35.4%-45.8%), respectively. The most effective antibiotics detected against the isolates were imipenem and amikacin with the lowest resistance rates of 0.2% (range: 0%-1.1 %) and 0.6% (range: 0%-3.2%), respectively. The frequency of positive Intll gene and class 1 integron gene cassettes were found as 25.8% (162/626) and 16.6% (104/626), respectively, whereas the frequency of positive intl2 gene II and class 2 integron gene cassettes were 5.1 % (32/626) and 3% (19/626), respectively. The lowest intll gene frequency was detected in the isolates from Kayseri (16.6%) and the highest in the isolates from Kahramanmaras (35.4%) provinces. While there was no intl2 gene in the isolates from Denizli and Kayseri, the highest frequency was 12.1 % in the isolates from Şanliurfa province. dfrA1 gene, the most frequent gene among integron gene cassettes was positive in 31 class 1 integron gene cassette alone, and positive with aadA1 gene in 18 class 1 integron gene cassettes. dfrA1 gene was positive with aadA1a just in one isolate. dfrA1 7 allele was positive in one isolate alone, in 28 isolates with aadA1, and in 15 isolates with aadAS. aadA1 gene was detected in four isolates. dfrA17-sat-aadA5 co-existence was detected among class 2 integron gene cassette in isolates from six provinces. dfrA1-sat-aadA1 was detected in one isolate from Ankara province and dfrA1 was detected in one isolate in Nigde province only. As a result, dfrA1 and aadA1 genes are the most common types of genes among class 1 and class 2 integron gene cassettes in E.coli isolated from urine cultures. It was concluded that high resistance against streptomycin (31.2%) and SXT (41.2%) supported the dissemination of integron-mediated genes dfr, sul1 and aad in the isolates

Three cases of fungemia caused by Blastoschizomyces capitatus

Blastoschizomyces capitatus özellikle bağışıklık sistemi baskılanmış kişilerde ciddi ve fatal seyreden sistemik enfeksiyonlara neden olabilmektedir. B.capitatus suşlarının hastane enfeksiyonuna neden olduğu ve salgınlar yaptığı da bildirilmektedir. Bu raporda, hematolojik malignensisi olan üç hastada B.capitatus suşlarının neden olduğu fungemi olguları sunulmaktadır. Sunulan birinci olgu akut lenfoblastik lösemi tanısı alan 20 yaşında kadın hasta, ikinci olgu B hücreli malign lenfoma tanısı alan 26 yaşında kadın hasta ve üçüncü olgu B hücreli akut lenfoblastik lösemi tanısı alan 7 yaşında erkek hastadır. Olguların tümü kemoterapi almakta olup, nötropeni nedeniyle antibakteriyel ve antifungal ilaç tedavisi uygulanan hastalardır. Ampirik kaspofungin tedavisi altında iken ikinci ve üçüncü olguların kan kültüründe B.capitatus üremesi olmuş, etkenin tanımlanmasından sonra bu hastalara vorikonazol ve amfoterisin B tedavisi başlanmış ve takiplerinde iyileşme olduğu belirlenmiştir. Yine ampirik kaspofungin tedavisi alan birinci hasta ise, kan kültüründe B.capitatus üremesi saptanmadan önce kaybedilmiştir. Suşların tanımlanmasında konvansiyonel mikolojik yöntemler [mikroskobik ve makroskobik morfoloji, germ tüp testi, üre hidrolizi, karbonhidrat asimilasyon testleri (API 20C AUX; BioMerieux, Fransa), üreme ısısı ve sikloheksimid duyarlılığı] kullanılmış ve izolatlar annellokonidya oluşturma, üreaz negatifliği, karbonhidrat kullanımı, 45°C’de üreme ve sikloheksimide dirençli olmaları ile B.capitatus olarak tanımlanmıştır. Suşların antifungal duyarlılığı, mikrodilüsyon yöntemi (amfoterisin B, vorikonazol, itrakonazol, flukonazol, ketakonazol) ve E-test (kaspofungin) ile araştırılmıştır. Çalışmada, üç B.capitatus izolatının minimum inhibitör konsantrasyon (MİK) değerleri amfoterisin B için sırasıyla 0.25, 0.125, 0.032 ?g/ml; flukonazol için 2, 2, 16 ?g/ml; itrakonazol için 0.064, 0.032, 0.032 ?g/ml; ketokonazol için 0.125, 0.064, 0.064 ?g/ml; vorikonazol için tümünde 0.032 ?g/ml ve kaspofungin için tümünde > 32 ?g/ml olarak bulunmuştur. Olguların kan kültürlerinden B.capitatus izolasyonu yaklaşık 15 gün içinde -ard arda- yapılmış olduğundan, benzerlik oranının araştırılması için suşların genotipi “repetitive sequence-based PCR” (DiversiLab System; BioMerieux, Fransa) yöntemiyle belirlenmiştir. Analiz sonucunda benzerlik oranı suşların ikisinde (1. ve 2. olgu) %97, birinde (3. olgu) %94.9 olarak bulunmuş; ancak çevresel örnekler alınmadığından bulaş şekli açıklık kazanmamıştır. Sonuç olarak, immün yetmezlikli hastalarda sistemik fırsatçı mantar enfeksiyonlarında B.capitatus’un da akılda tutulması gerekmektedir. Klinik B.capitatus suşlarının in vitro antifungal duyarlılıklarının belirlenmesi, tedavi yaklaşımlarına ve epidemiyolojik verilere katkı sağlayacaktır.Blastoschizomyces capitatus is a rare fungal pathogen that may lead to severe and fatal systemic infections especially in immunosuppressive individuals. B.capitatus strains have also been reported as the cause of hospital-acquired infections and outbreaks. In this report, three fungemia cases caused by B.ca- pitatus with hematologic malignancies have been presented. The first case was a 20-year-old female with acute lymphoblastic leukemia, the second was a 26-year-old female with B-cell malignant lymphoma and the third was a 7-year-old male with B-cell acute lymphoblastic leukemia. All of the patients have been receiving chemotherapy, and treated with antibacterial and antifungal agents due to neutropenia. The blood cultures obtained from the second and third patients yielded B.capitatus although they were under empirical caspofungin therapy. Those patients have been treated with voriconazole and ampho- tericin B after the identification of B.capitatus, and clinical improvement were noted during their follow- up. However the first patient who was also under caspofungin therapy had died just before the isolation of B.capitatus from her blood culture. Conventional mycological methods [macroscopic and microscopic morphology, germ tube test, urea hydrolysis, carbohydrate assimilation tests (API 20C AUX; Bi- oMerieux, France), growth temperature, cycloheximide sensitivity] were used for the identification of the isolates. The strains were identified as B.capitatus with the characteristics of annelloconidia formation, urease negativity, carbohydrate utilization, growth at 45ºC and resistance to cycloheximide. Antifungal susceptibilities of isolates were determined by using microdilution method (for amphotericin B, fluconazole, itraconazole, voriconazole, ketoconazole) and E-test (for caspofungin). Minimum inhibitory concentration (MIC) values of the three B.capitatus strains were detected as 0.25, 0.125, 0.032 µg/ml for amphotericin B; 2, 2, 16 µg/ml for fluconazole; 0.064, 0.032, 0.032 µg/ml for itraconazole and 0.125, 0.064, 0.064 µg/ml for ketoconazole, respectively, while MIC values of all strains were 0.032 µg/ml for voriconazole and > 32 µg/ml for caspofungin. Since B.capitatus strains were isolated from the cases within about 15 days -sequentially-, the genotypes of the isolates were determined by repetitive sequence- based PCR (DiversiLab System; BioMerieux, France) to investigate the similarity rates. The results of analysis indicated 97% similarity between two (case 1 and 2) strains and 94.9% similarity in one strain (case 3) of B.capitatus, however the transmission route could not be clarified due to the absence of environmental sampling. In conclusion, B.capitatus should also be considered as a cause of systemic fun- gal infections in immunocompromised patients. Determination of the in vitro antifungal susceptibilities of clinical B.capitatus strains may contribute to the therapeutic approaches and epidemiological data

Characterization of novel VIM carbapenemase, VIM-38, and first detection of GES-5 carbapenem-hydrolyzing beta-lactamases in Pseudomonas aeruginosa in Turkey

Pseudomonas aeruginosa isolates were collected form a Turkish hospital. Antimicrobial susceptibility was performed using the Vitek 2 Compact system, and 24 isolates were categorized as multidrug resistant (n = 18), extensively-drug resistant (n = 5), or pan-drug resistant (n = I). PCR and DNA sequence analysis revealed that 1 strain possessed the bla(GES-5) and another carried a novel bla(VIM) variant, named VIM-38. This new gene exhibited 1 amino acid substitution (Ala265Val) in comparison to its closest variant, VIM-5. Both VIM encoding genes were clones and demonstrated similar susceptibility profile when expressed in identical background. The presence of VIM-38 increases the diversity of carbapenemases in Turkey. (C) 2014 Elsevier Inc. All rights reserved.This work was supported by Recep Tayyip Erdogan University Research Fund Grants BAP-2012.106.01.11 and BAP-2011.102.03.3