Atık sularda genişlemiş spektrumlu beta laktamaz sentezleyen escherichia coli'lerin varlığının araştırılması ve potansiyel halk sağlığı problemleri
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Tarih
2023
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Hatay Mustafa Kemal Üniversitesi
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info:eu-repo/semantics/openAccess
Özet
Bu çalışmada genişlemiş spektrumlu beta-laktamaz üreten E. coli'lerin (GSBL-EC) Hatay ilinde bulunan iki farklı şehir atık sularında varlığının ortaya koyulması ve elde edilen izolatların karakterizasyonu amaçlandı. Tez çalışması kapsamında, Hatay ilindeki iki atık su arıtma tesisinden (AAT; Antakya ve İskenderun) 3 ay boyunca 24 adet su numunesi (giriş (n=12) ve çıkış (n=12)) toplandı. Toplanan su numuneleri seri şekilde seyreltilerek seftazidim içeren Tripton-safra X-glukuronid agara (TBX) yayıldı ve 24 saatlik inkubasyon süresinden sonra koloni sayımı yapıldı. Mavi-yeşil koloniler seçilerek bu kolonilerin kanlı agara ekimleri yapıldı. Elde edilen izolatların tür düzeyinde identifikasyonu uspA geni kullanılarak polimeraz zincir reaksiyonu (PZR) ile belirlendi. Tüm izolatlarda uspA geninin varlığı tespit edildi. İzolatların GSBL doğrulamaları, çift disk sinerji yöntemi ve kombine disk yöntemi ile yapıldı. İzolatların klonal yakınlıkları ise darbeli alan jel elektroforezi (PFGE) kullanılarak belirlendi. PFGE analizde bir izolat test edilemedi. Kalan 65 izolatın bant profilleri incelendiğinde ise (eşik değer >%85), 16 izolatın klonal olarak bağlantılı olduğu ve 49 izolatın ise bağlantılı olmadığı gözlemlendi. İzole edilen GSBL-EC izolatlarının farklı antimikrobiyallere karşı direnç durumları belirlendi. GSBL-EC izolatlarının penisilin, seftazidim ve sefalotine %100 dirençli olduğu, imipeneme karşı ise herhangi bir direnç bulunmadığı tespit edildi. Elde edilen tüm GSBL-EC izolatlarının, beta-laktam, dezenfektan ve kinolon direnç genleri PZR ile belirlendi. İzolatların %86,36'sında (n=57) blaCTX-M geni bulundu ve Sanger sekanslama sonucuna göre %89,47 blaCTX-M-15, %1,75 blaCTX-M-1, %7,01 blaCTX-M-55 ve %3,5 blaCTX-M-3 olarak belirlendi. Ayrıca, izolatların %54,4'ünde (n=36) blaTEM geni ve %7,57'sinde (n=5) blaCMY-2 geni tespit edilirken, hiçbir izolatta blaSHV genine rastlanılmadı. Kinolon direnç genleri sırasıyla qnrA1 (%3,03; n=2), qnrB1 (%4,54; n=3) ve aac(6)-Ib (%10,60; n=7) oranlarında bulundu. qnrC ve qnrD genleri ise izolatların hiçbirinde tespit edilmedi. İzolatlarda dezenfektan direncine aracılık eden genler ise sırasıyla ydgE (%84,84; n=56), qacEΔ1 (%81,81; n=54), ydgF (%80,30; n=53), mdfA (%33,33; n=22), emrE (%30,30; n=20), qacF/H/I (%7,57; n=5), sugE(c) (%6,06; n=4) ve sugE(p) (%3,03; n=2) olarak bulundu, buna karşın oqxA geni hiçbir izolatta tespit edilmedi. Sonuç olarak, Antakya'daki AAT-Çıkış (AAT-E)'den alınan örnekleme noktaları, İskenderun'daki AAT-E'den daha fazla GSBL-EC yükünün olduğunu gösterdi. Her iki AAT'nin çıkış suyu örneklerinde blaCTX-M-15 genini taşıyan E. coli'nin yaygın bir şekilde bulunması halk sağlığı açısından önemli bulundu.
The aim of this study was to determine the presence of extended spectrum beta-lactamase producing E. coli (ESBL-EC) in wastewater from two different cities in Hatay province and to characterize the isolated strains. Within the scope of the thesis study, 24 water samples (influent (n=12) and effluent (n=12)) were collected from two wastewater treatment plants (WWTP; Antakya and Iskenderun) in Hatay province for 3 months. The collected water samples were serially diluted and spread on Tryptone Bile X-glucoronide Agar (TBX) containing ceftazidime and colony counts were performed after 24 hours of incubation. Suspicious blue-green colonies were selected and these colonies were inoculated on blood agar. Species level identification of the isolates obtained was determined by polymerase chain reaction (PCR) using the uspA gene. Presence of uspA gene was detected in all isolates. ESBL confirmation of the isolates was performed by double disk synergy method and combined disk method. Clonal affinity of the isolates was determined by pulsed field gel electrophoresis (PFGE). One isolate could not be tested in PFGE analysis. When the band profiles of the remaining 65 isolates were analysed (cut-off >85%), 16 isolates were clonally related and 49 isolates were not. The resistance status of a total of 66 ESBL-EC isolates against different antimicrobials was determined. ESBL-EC isolates were found to be 100% resistant to penicillin, ceftazidime and cephalothin, while there was no resistance to imipenem. Beta-lactam, quinolone and disinfectant resistance genes of all ESBL-EC isolates were determined by PCR. The blaCTX-M gene was found in 86,36% (n=57) of the isolates and according to Sanger sequencing results, 89,47% were blaCTX-M-15, 1,75% blaCTX-M-1, 7,01% blaCTX-M-55 and 3,5% blaCTX-M-3. In addition, blaTEM gene was detected in 54.4% (n=36) and blaCMY-2 gene was detected in 7,57% (n=5) of the isolates, while no blaSHV gene was found in any isolates. Quinolone resistance genes were found in qnrA1 (3,03%; n=2), qnrB1 (4,54%; n=3) and aac(6)-Ib (10,60%; n=7), respectively. qnrC and qnrD genes were not detected in any of the isolates. The genes mediating disinfectant resistance in isolates were ydgE(84,84%; n=56), qacEΔ1 (81,81%; n=54), ydgF (80,30%; n=53), mdfA (33,33%; n=22), emrE (30,30%; n=20), qacF/H/I (7,57%; n=5), sugE(c) (6,06%; n=4); n=22), and sugE(p) (3,03%; n=2), whereas oqxA gene was not detected in any isolate. In conclusion, sampling points from WWTP-Effluent (WWTP-E) in Antakya showed a higher ESBL-EC load than WWTP-E in Iskenderun. As a result, the sampling points taken from WWTP-E in Antakya showed a higher ESBL-EC load than WWTP-E in Iskenderun. The widespread presence of E. coli carrying the blaCTX-M-15 gene in the effluent samples of both WWTPs was found to be important for public health.
The aim of this study was to determine the presence of extended spectrum beta-lactamase producing E. coli (ESBL-EC) in wastewater from two different cities in Hatay province and to characterize the isolated strains. Within the scope of the thesis study, 24 water samples (influent (n=12) and effluent (n=12)) were collected from two wastewater treatment plants (WWTP; Antakya and Iskenderun) in Hatay province for 3 months. The collected water samples were serially diluted and spread on Tryptone Bile X-glucoronide Agar (TBX) containing ceftazidime and colony counts were performed after 24 hours of incubation. Suspicious blue-green colonies were selected and these colonies were inoculated on blood agar. Species level identification of the isolates obtained was determined by polymerase chain reaction (PCR) using the uspA gene. Presence of uspA gene was detected in all isolates. ESBL confirmation of the isolates was performed by double disk synergy method and combined disk method. Clonal affinity of the isolates was determined by pulsed field gel electrophoresis (PFGE). One isolate could not be tested in PFGE analysis. When the band profiles of the remaining 65 isolates were analysed (cut-off >85%), 16 isolates were clonally related and 49 isolates were not. The resistance status of a total of 66 ESBL-EC isolates against different antimicrobials was determined. ESBL-EC isolates were found to be 100% resistant to penicillin, ceftazidime and cephalothin, while there was no resistance to imipenem. Beta-lactam, quinolone and disinfectant resistance genes of all ESBL-EC isolates were determined by PCR. The blaCTX-M gene was found in 86,36% (n=57) of the isolates and according to Sanger sequencing results, 89,47% were blaCTX-M-15, 1,75% blaCTX-M-1, 7,01% blaCTX-M-55 and 3,5% blaCTX-M-3. In addition, blaTEM gene was detected in 54.4% (n=36) and blaCMY-2 gene was detected in 7,57% (n=5) of the isolates, while no blaSHV gene was found in any isolates. Quinolone resistance genes were found in qnrA1 (3,03%; n=2), qnrB1 (4,54%; n=3) and aac(6)-Ib (10,60%; n=7), respectively. qnrC and qnrD genes were not detected in any of the isolates. The genes mediating disinfectant resistance in isolates were ydgE(84,84%; n=56), qacEΔ1 (81,81%; n=54), ydgF (80,30%; n=53), mdfA (33,33%; n=22), emrE (30,30%; n=20), qacF/H/I (7,57%; n=5), sugE(c) (6,06%; n=4); n=22), and sugE(p) (3,03%; n=2), whereas oqxA gene was not detected in any isolate. In conclusion, sampling points from WWTP-Effluent (WWTP-E) in Antakya showed a higher ESBL-EC load than WWTP-E in Iskenderun. As a result, the sampling points taken from WWTP-E in Antakya showed a higher ESBL-EC load than WWTP-E in Iskenderun. The widespread presence of E. coli carrying the blaCTX-M-15 gene in the effluent samples of both WWTPs was found to be important for public health.
Açıklama
Anahtar Kelimeler
Mikrobiyoloji, Microbiology
