Osman Birol ÖZGÜMÜŞ, Murat ERTÜRK, Faruk AYDIN, İlknur TOSUN, Ali Osman KILIÇ

Carriage of mobilizable plasmid-mediated beta-Lactamase gene in ampicillin-resistant Escherichia coli strains with origin of normal fecal flora

Amaç: Bu çalışmanın amacı, insanın normal fekal florasından izole edilen ampisiline dirençli (Ampr) Escherichia coli izolatlarında β-laktamaz genlerinin taşıyıcılığının araştırılmasıydı. Yöntemler: En az üç ay boyunca antibiyotik kullanmamış 21 sağlıklı kişinin dışkı örneklerinden izole edilen 10 Ampr E. coli suşu TEM-, SHV-, ve OXA-tipi β-laktamaz genleri açısından polimeraz zincir reaksiyonu (PZR) ile tarandı. Suşların antibiyotiklere hassasiyetleri disk difüzyon metodu ile, ampisilinin suşlara karşı minimum inhibitör konsantrasyonu (MİK) agar sulandırım metodu ile belirlendi. Plazmit aktarma deneyleri sıvıda çiftleşme metodu ile yapıldı. Plazmit DNA'sı alkali lizis tekniği ile izole edildi. Hibridizasyon deneylerinde digoxygenin ile işaretli TEM-1 probu kullanıldı. Bulgular: On suşun ikisinin PZR ile yalnızca TEM-tipi geni (blaTEM) taşıyıcısı olduğu bulundu ve ampisiline karşı dirençleri konjugatif olarak bir rekombinant E. coli K-12 suş C600'e aktarıldı. Ampisilinin iki orijinal suşa ve transkonjugantlarına karşı MİK'leri >512 µg/mL olarak tespit edildi. Ayrıca, bu iki suş ve transkonjugantlarında β-laktamaz inhibitör direnci de gözlendi. Digoxygenin ile işaretli TEM-1 DNA probu TEM-geni taşıyan iki organizmadan izole edilmiş olan bazı non-konjugatif fakat taşınabilir plazmit DNA'larına hibridize oldu. Sonuç: Bu sonuçlar; barsak ortamında β-laktamaz geni taşıyan kommensal E. coli suşlarının direnç determinantlarını küçük direnç plazmitleri (R plazmit) üzerinde taşıyabildiğini ve yakın zamanda antibiyotik kullanılmasa dahi bunların toplumda direnç genlerinin potansiyel bir rezervuarı haline gelebileceğini göstermektedir.

Normal fekal flora kökenli ampisiline dirençli Escherichia coli suşlarında taşınabilir plazmit aracılı beta-laktamaz gen taşıyıcılığı

Aim: The aim of this study was to investigate the carriage of β-lactamase genes in ampicillin-resistant (Ampr) Escherichia coli (E. coli) isolates from human normal fecal flora. Methods: Ten Ampr E. coli strains isolated from the stool samples of 21 healthy persons with no antibiotic use during at least three months were screened for TEM-, SHV-, or OXA-type β-lactamase genes by polymerase chain reaction (PCR). The susceptibility of the strains to antibiotics was determined by disk diffusion method, and minimum inhibitory concentration (MIC) of ampicillin to the strains was determined by agar dilution method. Plasmid transfer assays were performed by broth mating technique. Plasmid DNA was isolated by alkaline lysis method. Digoxigenin-labeled TEM-1 probe was used in hybridization assays. Results: Two of 10 strains were found to be carrier for only TEM-type β-lactamase gene (blaTEM) by PCR, and their resistances to ampicillin were conjugatively transferred to a recombinant E. coli K-12 strain C600. MIC of ampicillin to two representative strains and their transconjugants was detected as >512 µg/ml. Moreover, β-lactamase inhibitor resistance was also observed in these two strains and their transconjugants. Digoxigenin-labeled TEM-1 DNA probe was hybridized to some non-conjugative but mobilizable plasmid DNAs purified from two of the TEM-gene-carrying organisms. Conclusions: These results indicate that commensal E. coli strains carrying β-lactamase gene in the bowel environment could retain resistance determinants on small-sized resistance plasmids (R plasmids) and become a potential reservoir for resistance genes in the community, even in the absence of recent antibiotic consumption.

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