Shahid Beheshti University of Medical Sciences,Tehran,Iran Tabriz University of Medical Science, Iran

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2 Shahid Beheshti University of Medical Sciences,Tehran,Iran Tabriz University of Medical Science, Iran 2

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4 میکروارگانیسم عامل های عفونت بیمارستانی دلیل به انتی به باال مقاومت مشکالت ها بیوتیک برای را بزرگی بیماران و بخش درمانی ایجاد می کنند. بعضی در کشورها میزان های عفونت بیمارستانی دالیل به فقدان نظارت عدم عفونت از پیشگیری کاربرد نامناسب انتی بیوتیک ها و ازدحام بیش از حد بیماران باال می باشد. در بین باک تری ها اسینتوباکتر بامانی یک پاتوژن مهم بیمارستانی با ايجاد باکتریمی عفونت های تنفسی مجاری ادراری و... در بیمارستان ها در نقاط مختلف دنیا شناخته شده است که کنترل عفونت های حاصل از ان به علت مقاومت های داروئی چندگانه مشکل است. مسئله این درمان داروئی وضعیت در بیماران بخش در ویژه به را بحرانی دشواری با ویژه های مراقبت می رو روبه کند. 4

5 مورفولوژی و خصوصيات رشد کوکوباسيل مورف پلی منفی گرم های متابوليسم فاقد تخميری اجباری هوازی کپسولدار مثبت کاتاالز منفی اکسيداز منفی اندول رشد توانایی در 42 درجه بدون حرکت و اسپور بدون پيگمان بدون خوندار محيط در ها كلنی موکوئيدی تا صا ف هموليز دارای در شرایط بقا به قادر نامساعد برای مدت طوالنی

6 virulence Factors Survive under dry & iron-deficient conditions 1/3 Lipopoly saccharid capsule Outer membrane protein A Two component regulatory system Fimbriae (adhere to human bronchial epithelium) Biofilm associated protein penicillin-binding protein Phosphplipase D Quorum sensing 6

7 Environmental reservoirs Soil, fresh water, vegetables, animals Survives in dry particles and dust for up to 10 days (7 days for S. aureus) Encapsulated strains survive for >4 months on PVC, ceramics, rubber, steel (inanimate objects) Survives exposure to chlorhexidine, gluconate and phenolbased disinfectants Survives exposure to radiation Difficult to eradicate

8 مکانیسم های مقاومت انتی بیوتیکی دراسینتوباک تر پمپ های ترشحی تغییر هدف انتی بیوتیک کاهش جذب انتی بیوتیک غیرفعال کردن انزیمی.a.b.c.d سویه های مقاوم :(MDR) سويه هاي مقاوم به حداقل سه كالس انتي بيوتيك 8

9 Bacterial Efflux Pumps Responsible for extrusion of toxic substances and antibiotics outside the cell. classified into five major superfamilies, based on the amino acid sequence and the energy source, RND and MATE systems are the most prevalent in multiply resistant A. baumannii. adeabc, RND pumps typically exhibit a wide substrate range that can include antibiotics, dyes, biocides, detergents, and antiseptics. The major antibiotic classes that effluxed by the RND-type pumps are macrolides, Aminoglicosides, fluoroquinolones, TMP and fourthgeneration, cephems. 9

10 Bacterial Efflux Pumps AbeM, that belongs to the MATE family has also been characterized in isolates of A. baumannii. Substrates for the AbeM efflux pump include Gentamicin, Ciprofloxacin, Erythromycin, and Trimethoprim. 10

11 Efflux Pumps antibiotic efflux pump EP inhibitor bacteria cell membrane antibiotic target 11

12 Treatment A.baumanni is one of the Red Alert pathogens A. very difficult to treat, combination therapy is effective treatment: Aminoglycosides with an effective B-lactam, B lactam with either a fluoroquinolone or rifampin. Colistin with rifampin or Meropenem treatment failure and death caused by Acinetobacter infections or underlying diseases are common. 12

13 æ Antibiotic susceptibility test by Kirby- Bauer disc diffusion according to CLSI guidelines æ MIC determination by E-test method according to CLSI guidelines æ Detection of oxa 51 like gene by PCR æ Detection of adea, adeb, adec and abem genes by PCR and Sequencing methods

14 Materials and Methods 14

15 Bacterial isolates From June 2013 to march 2014 sixty one isolates of A. baumannii Imam Reza hospital in Tabriz, Iran The isolates were identified by conventional biochemical tests The isolates were stored at c in trypticase soy broth (TSB) containing % glycerol

16 جمع آوری نمونه انواع ادرار زخم خون مایعات استریل ترشحات ریوی نمونه ها خلط آبسه محل جمع آوری بيمارستان امام رضا )ع ) 16

17 تست های بيوشيميایی برای تشخيص گونه اسينتوباکتر 1. تست های بيوشيميایی 2. بررسی وجود آنزیم کاتاالز 4. تست اکسيداز. 3 رشد در دمای 42 C 17

18 Susceptibility testing imipenem (10 μg), meropenem (10 μg) ceftazidime (30 μg), cefotaxime (30 μg), ciprofloxacin (5 μg), cefepime (30 μg), ceftriaxon (30 μg) trimethoprim-sulfamethoxazole (2.5 μg), amikacin (AN: 30 μg), tobramicin (TOB: 10 μg ) and gentamicin (10 μg) colistin (10 μg) Escherichia coli ATCC25922 was used as a control strain 18

19 Minimum Inhibitory Concentration (MIC) E- test method (AB Biodisk, Solna, Sweden) according to CLSI guidelines 2012 Imipenem: to 32 μg/ml Amikacin: to 256 μg/ml ciprofloxacin: to 32 μg/ml control strain: Escherichia coli ATCC

20 adea, adeb, adec, abem and OXA-51 Like genes Extraction Kit (Bionner Company,Cat. No. K ). 20

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22 Primer Sequence (5-3 ( Gene Product size (bp) adea -F CTGATATTACAGGGGTGTG adea 408 adea -R GCTTCTCTCAATAAAGCTGAAG adeb -F adeb -R ATTTGGATTGCTGAGCATTC GTAAACCTTGCTGACGTACA adeb 340 adec -F adec -R ATGCATCATCTGAACTGAAAG GTGCATGTGTAGCAAGTGCA adec 222 abem F abem-r TATTACTTACCTTGCAACGCAG GTGGTTGCAATCATGATGCCA abem

23 Materials Concentration volume Buffer 10 X 1 X 2.5 μl MgCl 2 25 mm Primer (F, R) Taq pol 5 u/μl 2.5 mm 0.5μM 1.5 U 0.8 μl 1μl 0.2 μl dntp Mix 2mM 0.2 mm 0.5 μl DNA Template DW 15μg/ml - 1 μl 14 μl Final volume - 20 μl 23

24 Electrophoresis æ æ æ Horizontal electrophoresis 1% agarose gel 50 bp (100 bp) DNA ladder as marker æ TBE 1X (Tris-boric acid-edta ) æ æ Gel staining with ethydium bromide uv transluminator 24

25 Sequencing sequencing was performed by the Bioneer Company (Korea). The nucleotide sequences were analysed with Chromas 1.45 software and BLAST in NCBI. 25

26 Results

27 The Frequency distribution of patients with A.baumannii in Imam Reza hospital based on Sex 27

28 The Frequency & Percentage of patients with A.baumannii in Imam Reza hospital based on age 28

29 Distributions of Acinetobacter baumannii isolates in samples 29

30 Susceptibility testing 30

31 Distributions of 61 samples based on antibiotic resistance 31

32 Minimum Inhibitory Concentration (MIC) 32

33 Results of MIC test using E-test imipenem:73.3%, ciprofloxacin: 93.3% and amikacin: 38%. 33

34 Absolute & relative frequency of Efflux pump genes by PCR method 34

35 Detection of Oxa 51gene in A. baumannii by PCR On 353bp 500 bp 353bp 35

36 Detection of adea gene in A. baumannii by PCR On 408bp 500 bp 36

37 Detection of adeb gene in A. baumannii by PCR On 340 bp 500 bp 340 bp 37

38 Detection of AdeC gene in A. baumannii by PCR On 222 bp 500 bp 38

39 Detection of AbeM gene in A. baumannii by PCR On 283 bp 500 bp 39

40 Sequencing of adeb-14 gene > Ab14- adeb TTCCAGTGTGGCCCCGCCACAGGTGAATATTAGTGCGACTTATCCTGGTGCTACAGC TAAAACCATTAACGATAGCGTTGTAACCTTAATTGAGCGCGAATTATCGGGTGTAAA AAATCTACTCTACTATAGTGCGACAACAGATACCTCCGGTACAGCAGAGATTACGGC TACGTTTAAACCAGGCACAGATGTGGAAATGGCTCAGGTTGACGTTCAAAATAAAA TCAAGGCTGTAGAAGCTCGCTTACCGCAAGTTGTACGTCAGCAAGGTTTACAATG Electropherogram adeb-14 gene 40

41 Phylogenetic tree 41

42 42

43 43

44 The number of efflux pump gene sequences in GenBank access 44

45 45

46 The lowest rates of resistance in isolates were observed for Colistin 6 (11%), Amikacin 30 )48%) and Tobramicin 31 (5o%). So, the best coverage against the study isolates was obtained with Amikacin, Tobramicin and Colistin..

47 Name Year Location Strain MDR Imipenem Meropenem Bromand 2009 Tehran % 24.6 (E-test) Ferreia 2011 Brezil - 68% 79% Peymani 1391 Tabriz % 68% /69% Goudarzi 1392 Tehran % 92% Our study 1393 Tabriz % 98% 73.3 (E-test)

48 Yavari, 2013, Orumieh Detection of efflux pumps in drug resistance / desinfectent level 100% Michael J, 2012, Spania 100 sample hospital environmental; Clonization A. baumannii by RT- PCR 100% sensitivity Ming-Feng Lin: 2011; 5 proximal hospitals in Taiwan; 23 MDR A. baumannii samples. antibiotic resistance : 82.6 to 100%. PCR screening: inti1 (91%), blaoxa 23 (57%), ampc(100%), adeb (100%), adej (100%), and abem (100% ) genes. Li Lin: 2009 in China; 112 clinical isolates; frequency of RND (adesr-adeabc, adede and adeijk drug efflux system) genes by (PCR & RT-PCR) (75%) Our study: 2013 in Tabriz, frequency of RND & MATE: 88 to 100% 48

49 نتیجه گیری شیوع ژنهای مقاومت به انتی بیوتیک در این مطالعه موجب نگرانی است. فراوانی باالی ژن های پمپ افالکس در ایزوله های برگرفته از بیمارستان امام رضا )ع( تبریز نشاندهنده این مطلب است که یکی از مکانیسم های دخیل در ایجاد مقاومت باالی این ایزوله ها نسبت به انتی بیوتیک های امینوگلیکوزیدی سیپروفلوکساسین تتراسایکلین کارباپنم ها و بتاالکتام ها ممکن است پمپ های تراوشی adeabc باشد. کنترل برای اینرو از گسترش از جلوگیری و عفونت باک تریهای مدیریت به نیاز دارو به مقاوم در دقیق تجویز دارو و شناسای ی ایزوله های مقاوم می باشد.

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