Klinik materyallerin izole edilen stafilokoklarda Makrolid,Linkozamid ve Streptogramin B direnci sıklığının Disk Yaklaştırma Testi ve moleküler yöntemler kullanılarak araştırılması
Subject of treatise:investigation of resistance frequency to macrolid Lincozamide and Streptopramin B in clinically isolated staphyloccocci by using D-test and moleculer methods
Yazar: NERİMAN BALABAN
Danışman: PROF. SEMRA KUŞTİMUR
Gazi Üniversitesi / Sağlık Bilimleri Enstitüsü / Mikrobiyoloji Ana Bilim Dalı
Derece: Doktora, 2008
Konu: Mikrobiyoloji = Microbiology

Özet:
Klinik Materyallerden izole Edilen Stafilokoklarda Makrolid,Linkozamid ve Streptogramin B Direnci Sikhginin Disk Yakla§tirma Testive Molekuler Yontemler Kullanilarak Ara§tinlmasi.Bu gah§mada Kasim 2004 - Eylul 2007 tarihleri arasindalaboratuvanmiza gonderilen ge§itli klinik materyallerden izole edilen vehastahk etkeni oldugu du§unulen 731 adet stafilokok su§unda MLSB tipidireng fenotipik ve genotipik yontemlerle belirlenmi§ ve su§lar arasindaklonal ili§kinin olup olmadigi incelenmi§tir.Bu su§lann 254 (%34,7) tanesi S. aureus, 477'si (%65,3)koaguiaz negatif stafilokok (KNS) olarak tanimlanmi§tir. S. aureussu§lannin 1251 (% 49,2) metisiline direngli (MRSA), 129'u (% 50,8)metisiline duyarli (MSSA) bulunmu§tur. KNS'lerde ise 306 su§ta (%64,2)metisilin direnci (MR-KNS) g6rulmu§, 171 (% 35,8) su§ ise metisilineduyarli (MS-KNS) bulunmu§tur.VITEK 2 sistemi kullanilarak KNS su§lan tur duzeyindetanimlanmi§tir. En sik rastlanan turler S. hominis 211 (%44,2), S.epidermidis 140 (%29,4) ve S. haemolyticus 55 (%11,5) olarakbelirlenmi§tir.Fenotipik olarak eritromisin direngli (ER-R), klindamisinduyarli (CL-S) bulunan su§lara D-test yontemi uygulanarak direngfenotipleri belirlenmi§tir. MRSA su§lannin 66'si (%52.8) S fenotipi, 4'u(%3,2) MS fenotipi, 34'u (%27,2) iMLSB fenotipi, 20'si (%16) cMLSBfenotipi olarak, MSSA su§lannin ise 119'u (%92.2) S fenotipi, 2'si (%1,6)MS fenotipi, 6'si (%4,7) iMLSB fenotipi, 1'i (%0,8) cMLSB fenotipi olarakbelirlenmi§tir. KNS turlerinde ise bu oranlar sirasiyla 147 (%30,8), 109(%22,9), 98 (%20,5) ve 119 (%24,9)'dur. Bir su§ ise D test ilesiniflandinlamayan direng paterni gostermi§tir. Bu eritromisin duyarh,klindamisin orta duyarh bulunan bir MRSA su§udur.iMLSe fenotipi gosteren 138, cMLSB fenotipi gosteren 140 su§untamami, rastlantisal segilen 59 MS fenotipi gosteren su§ ve kontrol grubuigin rastlantisal olarak segilen 70 S tipi su§un direng genleri ( ermA, ermB,ermC, msrA, msrB genleri agisindan ) PCR yontemiyle belirlenmi§tir.iMLSB fenotipinden 11, cMLSB fenotipinden 16 ve MS fenotipinden 3su§un higbir direng geni ta§imadigi tespit edilmi§tir. ermB geni isegah§maya ahnan higbir su§ta bulunmami§tir.iMLSB fenotipi gosteren su§lardan MRSA'larda ermA geninin,KNS'lerde ise ermC geninin daha sik oldugu bulunmu§tur.CMLSB fenotipi gosteren su§lar arasinda ermA ve ermC genleri ensik KNS su§lannda gozlenmi§tir.MS fenotipli su§larda en sik msrA+msrB gen kombinasyonubelirlenmi§ ve bu genleri birlikte ta§ima oraninin KNS su§lannda faziaoldugu g6rulmu§tur.Fenotipik olarak eritromisin direnci sergileyen 30 su§ta ise PCR ilehigbir direng geni bulunamami§tir.S fenotipli su§larda ermA, ermC, ermA+ermC genleri bulunmu§tur.ermA geninin MSSA ve MRSA su§lannda, ermC geninin ise KNSsu§lannda fazia oldugu g6rulmu§tur. Test edilen 70 su§un 33'unde en azbir direng geni belirlenmi§, 37 su§ta ise higbir direng geni bulunamami§tir.Sonug olarak bu gah§mada MLSB direncinden sorumlu gen ve genkombinasyonlarimn ulkemizde gorulme oranlannin yiiksek oldugubulunmu§tur. Fenotipik olarak duyarh gorunen su§larda da direnggenlerinin belirlenmi§ olmasi direng tesbitinde fenotipik yontemlerinyetersiz oldugunu ve molekuler yontemlerle de dogrulunmasi gerekliliginigostermi§tir. Direng geni ta§iyan bu su§lar in vivo direnggeli§tirebileceginden tedavi ba§ansizhginin onlenmesinde bu bulguonemlidir. Qali§mamizda eritromisin direngli oldugu halde higbir direnggeni ta§imayan su§lann bulunmasi direngten sorumlu bilinmeyenmekanizmalann olabilecegini de gostermi§tir.Molekuler tiplendirme RAPD yontemi ile yapilmi§tir. Gerek KNS'lergerekse S.aureuslarda hakim bir RAPD tipi belirlenememi§tir.

Abstract:
Investigation of the presence of Macrolide Lincosamide andStreptogramin B resistance among the Staphylococcus strains isolatedfrom various clinical specimens by using D-test and molecular methods.In this study, the presence of Macrolide Lincosamide andStreptogramin B (MLSB) resistance among 731 Staphylococcus strainsisolated from various clinical specimens sent for culture to our laboratorybetween November 2004 and September 2007 was investigated byphenotypic and genotypic methods. Clonal relatedness of the strains wasalso determined.Two hundred fifty four (34,7%) of the 731 staphylococci wereidentified as S. aureus, while 477 (65,3%) were coagulase-negativestaphylococci (CNS). Among the S. aureus strains, 125 (49,2%) weremethicillin-resistant (MRSA), and 129 (50,8%) were methicillin-sensitive(MSSA). Three hundred six CNS isolates (64,2%) were methicillinresistant (MR-CNS), while 171 (35,8%) were methicillin-sensitive (MSCNS).CNS strains were identified to species level by using theVITEK 2 system. The most frequent species were S. hominis (211isolates, 44,2%), S. epidermidis (140 isolates, 29,4%) and S. haemolyticus(55 isolates, 11,5%).When the strains were found to be phenotypically resistant toerythromycin (ER-R) and sensitive to clindamycin (CL-S), resistantphenotypes were determined by using the D-test method. Sixty six(52.8%) of the MRSA strains expressed the S phenotype, 4 (3,2%) MSphenotype, 34'u (27,2%) iMLSB phenotype, and 20 (16%) cMLSBphenotype. Among MSSA strains, 119 (92.2%) were S phenotype, 2(1,6%) MS phenotype, 6 (4,7%) iMLSB phenotype, and one (0,8%) cMLSBphenotype. In the CNS group, the number of strains expressing thesehenotypes were 147 (30,8%), 109 (22,9%), 98 (20,5%) and 119 (24,9%),respectively. One MRSA isolate which was erythromycin-sensitive andClindamycin intermediate, carried a resistance phenotype which could notbe determined by the D-test method.All of the 138 strains harboring the iMLSB phenotype, and 140strains with the cMLSB phenotype, along with randomly picked 59 isolateswith the MS phenotype and 70 control isolates with the S phenotype wereinvestigated for the presence of MLSB resistance genes (ermA, ermB,ermC, msrA, and msrB) by polymerase chain reaction (PCR) analysis.None of the resistance determining genes were found to be present in 11isolates of the iMLSB phenotype group, 16 of the CMLSB phenotype groupand 3 of the MS phenotype group. None of the strains were found to carrythe ermB gene.MRSA strains with iMLSB phenotype carried exclusively the ermAgene, while ermC gene was more prevalent among CNS.Among the strains showing the cMLSB resistance phenotype, ermAand ermC genes were more prevalent in the CNS strains.In the strains with the MS phenotype, msrA+msrB genecombination was more prevalent and CNS strains were found to carry bothof these genes more frequently.In 30 strains which were phenotypically resistant to erythromycin,none of the erm or msr resistance genes were found by PCR.The strains with the S phenotype were found to carry ermA, ermC,ermA+ermC genes. ermA gene was more prevalent among MSSA andMRSA strains, while ermC gene was more prevalent among CNS strains.In the 33 strains among the evaluated 70 strains, at least one resistancephenotype was determined, while in 37 strains no resistance determininggene was found.As a result, we have found that the prevalence of the genes, eitheralone or in combination, responsible for the MLSB resistance was high inour country. The presence of resistance genes in phenotypically sensitiveisolates, make us believe that phenotypic methods may not be sensitiveenough to determine MLSB resistance in Staphylococcus strains, andresistance must be confirmed by molecular methods. These resistancegenecarrying phenotypically sensitive strains may show in-vivo resistanceand may lead to unsuccessfulness of the therapy. The resistant strainswithout the erm or msr resistance gene may carry infrequent resistancedeterminants or even other mechanisms responsible for resistancealthough not yet discovered.