بررسي الگوي مقاومت آنتي بيوتيكي و فراواني ژن هاي blaVIM و blaNDM در اسينتوباكتر بوماني هاي ايزوله شده از بيماران بستري شهر هاي اصفهان و شهركرد

The evaluation of antibiotic resistance pattern and frequency of blaVIM and blaNDM genes in isolated Acinetobacter baumannii from hospitalized patients in Isfahan and Shahrekord

زمينه و هدف در حال حاضر بتالاكتام‌ها به عنوان داروي انتخابي در درمان عفونت‌هاي اسينتوباكتر بوماني مقاوم به چند دارو استفاده مي‌گردد، هرچند مقاومت به اين عوامل رو به افزايش مي‌باشد. اين باكتري با مكانيسم‌هاي مختلف از جمله توليد بتالاكتامازها به اين داروها مقاومت نشان مي‌دهند. متالوبتالاكتاماز داراي تيپ‌هاي مختلفي مي‌باشند كه غالبا پلاسميدي بوده و در بين آن‌ها blaVIM و blaNDMاز همه بارزتر هستند. اين مطالعه با هدف بررسي مقاومت دارويي و فراواني ژن‌هاي blaVIM و blaNDM در اسينتوباكتر بوماني‌هاي ايزوله شده از بيماران شهر‌هاي اصفهان و شهركرد به روش مولكولي اجرا گرديد. روش كار اين مطالعه مقطعي برروي 500 نمونه باليني اعم از خون، زخم، ادرار و مجاري تنفسي جمع آوري شده از 3 بيمارستان شهر‌هاي اصفهان (الزهرا و كاشاني) و شهركرد (كاشاني) طي مدت يك سال از فروردين 1397 تا فروردين 1398 انجام گرديد. پس از جمع‌آوري نمونه‌ها با استفاده از روش‌هاي كشت و بيوشيميايي گونه اسينتوباكتر بوماني شناسايي گرديد. سپس تست حساسيت آنتي بيوتيكي بر روي اين ايزوله‌ها با روش ديسك ديفيوژن بر اساس دستور CLSI انجام گرديد. در نهايت براي بررسي فراواني ژن‌هاي blaVIM و blaNDM با استفاده از پرايمرهاي اختصاصي PCR انجام شد. يافته‌ ها مقاومت آنتي بيوتيكي در 100 ايزوله اسينتوباكتر بوماني به ترتيب مربوط به آنتي بيوتيك‌هاي: سفي پيم (97%)، سفترياكسون (95%)، آميكاسين (95%)، ايمي پنم (76%)، پيپراسيلين-تازوباكتام (70%)، مروپنم (69%)، جنتامايسين (63%)، توبرامايسين (56%)، تتراسايكلين (51%)، آمپي سيلين-سولباكتام (49%) و كمترين مقاومت مربوط به پلي ميكسين B به دست آمد. نتايج PCR نشان داد كه به ترتيب 17% و 20%، سويه ها حامل ژن‌هاي blaVIM و blaNDMمي باشند. نتيجه‌ گيري افزايش ميزان شيوع بيمارستاني اسينتوباكتر بوماني، لزوم طراحي برنامه هاي حفاظتي نظير كنترل عفونت‌هاي ايجاد شده در بخش مراقبت‌هاي ويژه را خاطرنشان مي‌كند. همچنين با استفاده از روش‌هاي مولكولي مي‌توان اين باكتري‌ها را شناسايي و ويژگي مقاومت آنتي بيوتيكي آن‌ها را تعيين كرد و متناسب با آن آنتي بيوتيك‌ها را تجويز نمود.

Acinetobacter baumannii has emerged as an opportunistic nosocomial pathogen that causes ventilator-associated pneumonia, bacteraemia, endocarditis, wound infections, meningitis and urinary tract infections in patients with serious underlying disease and immunocompromised patients, and in those undergoing prolonged hospitalization and surgical procedures involving long-term antimicrobial therapy. The majority of nosocomial A. baumannii isolates are resistant to different classes of antibiotic including cephalosporin's, penicillin's, carbapenem's, fluoroquinolones and aminoglycosides. Multidrug-resistant (MDR) A. baumannii isolates are a serious problem in healthcare settings worldwide, especially in Europe, Asia, Latin America and other areas. The majority of MDR strains have been isolated from adults attending intensive healthcare units and are associated with increased patient mortality and persistence of isolates in the hospital environment. Horizontal transfer of antibiotic resistance genes is thought to play a major role in the emergence and development of MDR strains. Treatment of infections caused by highly resistant isolates, especially MDR, XDR (extensively drug resistant) and PDR (pan drug resistant), can be difficult. Currently, beta-lactams are used as a selective drug in the treatment of multi-drug resistant Acinetobacter spp., although resistance to these agents is increasing. Metalo-beta-lactamases have different types that are often plasmid and among them blaVIM and blaNDM are the most prominent. The aim of this study was to evaluate the drug resistance and the frequency of blaVIM and blaNDM genes in isolated isolates from Isfahan and Shahrekord cities by molecular methods. Methods This cross-sectional study was performed on 500 clinical specimens including blood, ulcer, urine and respiratory tract collected from 3 hospitals in Isfahan (Alzahra and Kashani) and Shahrekord (Kashani) during one year from April 2018 to April 2019. After assembling samples, biochemical methods of Acinetobacter spp were identified. Susceptibility of isolates to the following antibiotics was examined using the disk diffusion method according to the Clinical and Laboratory Standards Institute guidelines. Multidrug resistance (MDR) was defined as resistance to at least one agent in three or more antimicrobial categories. The antimicrobial susceptibility test of the isolated organisms was completed by the disc diffusion method using the Kirby–Bauer technique. According to the recommendation of CLSI, all tests were performed on Mueller Hinton agar. The surface was lightly and uniformly inoculated by a cotton swab. Prior to inoculation, the swab stick was dipped into a bacterial suspension having visually equivalent turbidity to 0.5 McFarland standards. The swab stick was then taken out and squeezed on the wall of the test tube to discard extra suspension. Inoculated plates were incubated at 35°C for 24 h. On the next day, plates were read by taking measurements of zone of inhibition. Finally, DNA extraction from overnight cultures of A. baumannii isolates was performed according to the protocol provided with the QIAGEN DNA Mini kit (QIAGEN Inc., Valencia, CA). PCR primers were used to determine the frequency of blaVIM and blaNDM genes. The data were analyzed with SPSS version 17.0 software (SPSS, Inc., Chicago, IL). A chi-square test was used to determine the statistical significance of the data. A p<0.05 was considered significant. Results Antibiotic resistance in 100 isolates of A. baumannii was related to antibiotics: Cefimoimide (97%), Ceftriaxone (95%), Amikacin (95%), Imipenem (76%), Piperacillin-tazobactam (70%), Meropenem (69%), Gentamicin (63%), Tobramycin (56%), Tetracycline (51%), and Ampicillin-Sulbactam (49%) and lowest resistance to Poly-Mixin B were obtained. The PCR results showed 17 and 20%, of strains carrying blaVIM and blaNDM genes, respectively. The prevalence rate of blaNDM in this study was 20%, which is contrary to studies conducted by Tognim et al. In 2011 (55%) which show that the prevalence rate of blaNDM has been decreasing over time. This decrease can be seen in the prevalence of blaVIM. Conclusion Increase in the incidence of A. baumannii in hospital points to the need to design protective programs such as controlling infections in the intensive care unit. Also, using molecular methods, these bacteria can be identified and their antibiotic resistance characteristics are determined and appropriate antibiotics are prescribed.