Publication: Rutin laboratuvarımızda etken olarak izole edilen Pseudomonas Aeruginosa kökenlerinde, Metalo Beta Laktamaz varlığının fenotipik ve genotipik yöntemlerle saptanması ve elde edilen sonuçların karşılaştırılması
Abstract
Rutin laboratuvarımızda izole edilen Pseudomonas aeruginosa kökenlerinde imipenem direncinin oldukça yüksek olması nedeniyle, bu kökenlerde, imipenem direnci ile metalo beta laktamaz ( MBL ) varlığı arasındaki ilişkiyi araştırarak; genotipik ve fenotipik yöntemler arasındaki uyumu saptayıp, rutin laboratuvarımızda uygulanabilir en uygun tarama yöntemi konusunda yaklaşım sahibi olabilmeyi amaçladık. 2002 - 2003 yılları arasında rutin laboratuvarımızda etken olarak izole ettiğimiz P. aeruginosa kökenlerinin imipenem ve seftazidim duyarlılıkları agar dilüsyon ve disk difüzyon yöntemleriyle çalışılmış ve elde edilen sonuçlar otomatize sistem sonuçlarıyla karşılaştırılmıştır. Otomatize sistemlerle seftazidime dirençli olduğu belirlenen 46 kökenin 8'i agar dilüsyonla, 7'si disk difüzyonla; aynı şekilde otomatize sistemlerle imipeneme dirençli olduğu belirlenen 59 kökenin 4'ü agar dilüsyonla, 3'ü disk difüzyonla duyarlı bulunmuştur. Agar dilüsyonla imipenem ve seftazidime duyarlı bulunan kökenler otomatize sistemlerle de duyarlı bulunmuştur. Otomatize sistemlerin kullanıldığı rutin laboratuvarlarda imipenem ve seftazidime direnç saptandığında, bu sonuçların diğer yöntemlerle ( özellikle dilüsyon yöntemleri ) doğrulanması gereklidir. İzole edilen kökenlerde metalo beta laktamaz varlığı fenotipik ve genotipik yöntemlerle araştırılmıştır. PCR sonuçları kriter olarak alınıp, fenotipik sonuçların güvenilirliği değerlendirilmiştir. Buna göre; hiçbir kökenimizde bla IMP ve bla VIM genlerine rastlanmadığından, fenotipik yöntemlerin duyarlılıkları yorumlanamamış olup, özgüllükleri irdelenmiştir. Çalışmamızda özgüllüğünü en yüksek bulduğumuz testler, CAZ-2-MPA çift disk sinerji yöntemi ( % 99 ) ve dört disk sinerji yöntemidir ( CAZ-2-MPA: % 100; FEP-2-MPA: % 94; CD-2-MPA: % 99 ). Fenotipik değerlendirmelerde, 2-MPA' nın EDTA ya göre daha özgül sonuç verdiği gözlenmiştir. Çok ucuz yöntemler olmalarına rağmen 2-MPA' nın kötü kokması ve kanserojen bir madde olması, testlerin PCR' a kıyasla hızlı sonuç vermemesi dezavantajlarıdır. Bunun yanı sıra, ticari olarak satılan E test MBL striplerininde özgüllüğünün ( % 47 ) oldukça düşük olduğu gözlenmiştir. Bizim ülkemizde MBL enzimine bağlı direnç, bugün için bir salgın riski ifade etmemektedir. Bu nedenle MBL enziminin varlığını her kökende fenotipik veya genotipik olarak incelemek hem iş yükünü hem de laboratuvar maliyetini arttıracağından, belirli aralıklarla rutin olarak izole edilen kökenlerde bu dirençten sorumlu gen bölgelerinin moleküler yöntemlerle gösterilmesinin uygun olacağını düşünmekteyiz. DEDECTİON AND COMPAİRE OF METALLO BETA LACTAMASE WİTH PHENOTYPIC AND GENOTYPIC METHODS IN PSEUDOMONAS AERUGINISA STRAINS ISOLATED IN ROUTINE MICROBIOLOGY LABORATORY.
Due to high isolation rate of imipenem resistant Pseudomonas aeruginosa strains in our hospital, we aimed to investigate the relationship between the resistance and metallo-beta-lactamese ( MBL ) production in those strains. To do so we compared the genotypical and phenotypical methods for MBL detection in order to develop the most suitable screening method for routine laboratorias. Imipenem and ceftazidime sensitivity of P. aeruginosa strains isolated in routine laboratory during a period from 2002 to 2003 were investigated using agar dilution and disk diffusion methods, and the obtained results were compared with the results of automated system. Of 46 strains determined to be resistant to ceftazidime by using automated systems, 8 were found to be sensitive with agar dilution and 7 with disk diffusion; in the same way, of 59 strains determined to be resistant to imipenem with automated systems, 4 were found to be sensitive with agar dilution and 3 with disk diffusion tests. The strains which were determined to be sensitive to imipenem and ceftazidime with agar dilution were all found to be sensitive with automatied system also. Therefore, when resistance to imipenem and ceftazidime is determined by using automated systems in routine laboratory, it is nec3essary to verify these results with standardized mthods ( especially with dilution methods ). In the isolated strains, the presence of metallo-beta-lactamese enzyme was investigated using genotypic and phenotypic methods. The results of PCR analysis were taken as criteria for evaluation of reliability of the phenotypic results. Accordingly, because bla IMP and bla VIM genes were not detected in any of our strains, the sensitivity of phenotypic methods could not be interpreted but the specificity was evaluated. In our study, the tests that were found to have the highest specificity were CAZ-2-MPA double disk synergy ( 99% ) and four disk synergy (CAZ-2-MPA: 100%; FEP-2MPA: 94%; CD-2-MPA: 99% ) methods. In phenotypic evaluations, 2-MPA was observed to give more specific results than EDTA. Although they are inexpensive methods, the bad odour and cancerogenic effects of 2-MPA and the time required the complete the procedures can be regarded as disadvantages when tests compared to PCR. In addition, the specificity ( 47 % )of the E test MBL strips was observed to be quite low. In our country, it seems likely that resistance due to MBL enzyme does not display an importent risk. Therefore, as it will be hard and costly to investigate presence of MBL enzyme genotypically and phenotypically in each isolate, we consider that it will be suitable to screen for genes responsible for this resistance in the routine isolate within given intervals.
Due to high isolation rate of imipenem resistant Pseudomonas aeruginosa strains in our hospital, we aimed to investigate the relationship between the resistance and metallo-beta-lactamese ( MBL ) production in those strains. To do so we compared the genotypical and phenotypical methods for MBL detection in order to develop the most suitable screening method for routine laboratorias. Imipenem and ceftazidime sensitivity of P. aeruginosa strains isolated in routine laboratory during a period from 2002 to 2003 were investigated using agar dilution and disk diffusion methods, and the obtained results were compared with the results of automated system. Of 46 strains determined to be resistant to ceftazidime by using automated systems, 8 were found to be sensitive with agar dilution and 7 with disk diffusion; in the same way, of 59 strains determined to be resistant to imipenem with automated systems, 4 were found to be sensitive with agar dilution and 3 with disk diffusion tests. The strains which were determined to be sensitive to imipenem and ceftazidime with agar dilution were all found to be sensitive with automatied system also. Therefore, when resistance to imipenem and ceftazidime is determined by using automated systems in routine laboratory, it is nec3essary to verify these results with standardized mthods ( especially with dilution methods ). In the isolated strains, the presence of metallo-beta-lactamese enzyme was investigated using genotypic and phenotypic methods. The results of PCR analysis were taken as criteria for evaluation of reliability of the phenotypic results. Accordingly, because bla IMP and bla VIM genes were not detected in any of our strains, the sensitivity of phenotypic methods could not be interpreted but the specificity was evaluated. In our study, the tests that were found to have the highest specificity were CAZ-2-MPA double disk synergy ( 99% ) and four disk synergy (CAZ-2-MPA: 100%; FEP-2MPA: 94%; CD-2-MPA: 99% ) methods. In phenotypic evaluations, 2-MPA was observed to give more specific results than EDTA. Although they are inexpensive methods, the bad odour and cancerogenic effects of 2-MPA and the time required the complete the procedures can be regarded as disadvantages when tests compared to PCR. In addition, the specificity ( 47 % )of the E test MBL strips was observed to be quite low. In our country, it seems likely that resistance due to MBL enzyme does not display an importent risk. Therefore, as it will be hard and costly to investigate presence of MBL enzyme genotypically and phenotypically in each isolate, we consider that it will be suitable to screen for genes responsible for this resistance in the routine isolate within given intervals.