Geniş spektrumlu beta laktamaz üreten ve üretmeyen klinik klebsiella pneumoniae suşlarında antibiyotik duyarlılıkları, blaCTX-M, blaTEM, blaOXA, blaSHV GSBL genlerinin ve PMQR genlerinin moleküler karakterizasyonu
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Tarih
2020
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Selçuk Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Klebsiella pneumoniae (K. pneumoniae), yoğun balgam üreten, enflamasyona ve akciğerlerde kalıcı hasarlara yol açan bakterilerdir. Pnömoniye ek olarak cerrahi girişim yapılan yerde enfeksiyona, idrar yolu enfeksiyonuna (İYE), ishale, üst solunum yolu enfeksiyonuna, yara enfeksiyonuna, menenjite, bakteriyemi ve septisemiye sebep olabilirler. Bakterinin kana bulaşması sonucu sepsis veya septik şok gelişir. Bu çalışmada, Konya ilindeki hastanelerdeki klinik materyallerden izole edilen (% 55,2 idrar) 192 adet K. pneumoniae suşlarının hem fenotipik hem de genotipik olarak GSBL üretim özelliklerinin, antibiyotik duyarlılıklarının belirlenmesi ve plazmit aracılı kinolon direnci (PMQR) genlerinin varlığının gösterilmesi amaçlanmıştır. GSBL üretim özellikleri fenotipik çift disk sinerji yöntemi ile belirlenmiştir. Antibiyotik duyarlılık testleri Bauer-Kirby tarafından önerilen disk difüzyon metodu ile yapılmıştır. Antibiyogram testi ve çift disk sinerji yöntemi için, 0,5 Macfarland yoğunluğunda hazırlanmış bakteri solüsyonları Mueller-Hinton agar yüzeyine yayılarak belli uzaklıklarda antibiyotik diskler yerleştirilmiştir. İnkübasyondan sonra zon çapları ölçülmüş ve dirançli/duyarlı suşlar belirlenmiştir. K. pneumoniae suşlarında genotipik olarak GSBL pozitiflerin belirlenmesi için, blaCTX-M, blaTEM, blaOXA, blaSHV beta-laktamaz genlerinin varlığı spesifik primer çiftleri kullanılarak multipleks PZR yöntemi ile taranmıştır. Tüm K. pneumoniae izolatlarında plazmit aracılı kinolon direncini ifade eden; qnrA (630 bç), qnrD (581 bç), qnrB (488 bç), qnrS (428 bç), oqxAB (313 bç), aac(6')-Ib-cr (260 bç), qepA (218 bç), qnrC (118 bç) genler spesifik primer çiftleri kullanılarak multipleks PZR ile taranmıştır. Çalışılan 192 K. pneumoniae izolatının 36 (%18.75)'sının fenotipik olarak, 186 (% 96.87)'sının genotipik olarak GSBL pozitif olduğu belirlenmiştir. K. pneumoniae şuşlarının antibiyogram sonuçlarına göre test edilen 19 antibiyotik arasında en yüksek direnç oranı % 97,9 ile Ampisiline karşı olduğu belirlenirken en düşük dirençe sahip antibiyotik ise Colistin (10mg) (%11,45) olmuştur. Suşların tamamının en az bir veya daha fazla antibiyotiğe dirençli olduğu ve suşlar arasında 113 farklı direnç profili olduğu görülmüştür. Çoklu antibiyotik direnç (ÇAD) indeksine göre tüm K. pneumoniae şuşlarının % 72 (139)'sinin direnç indeksinin 0,2'den yüksek olduğu belirlenmiştir. 192 K. pneumoniae suşunda GSBL genlerini görülme sıklığına göre en yaygın blaSHV geni içerdikleri 184 (% 95,83) suşta, ikinci sırada 95 (% 49,47) suşta blaTEM, üçüncü sırada blaOXA 87 (% 45,31) suşta, en az 27 (% 14,06) suşta blaCTX-M genini içerdikleri tespit edilmiştir. En yüksek % 26,5 oranında sadece blaSHV + blaTEM + blaOXA genlerinin birlikte bulunduğu GSBL gen kombinasyonuna rastlanmıştır. Tüm K. pneumoniae izolatlarının 8 PMQR genlerinin multipleks PZR sonucunda, en yaygın genin 156 şuşta rastlanan (% 81,25) oqxAB geni olduğu belirlenmiştir. En az rastlanan genin ise 7 suşta (% 3,6) qnrC geni olduğu ortaya konulmuştur. En yüksek % 21,87 oranında sadece oqxAB + aac(6')-Ib-cr + qnrD, genlerinin birlikte bulunduğu PMQR gen kombinasyonuna rastlanmıştır. Bu çalışma etkili tedavi için GSBL üreten suşların ve PMQR genlerinin tespit edilmesinin önemini ortaya koymuştur. Klinikte büyük problemlerden biri olan gereksiz ve yanlış antibiyotik kullanımının önüne geçilmesini sağlayabilmek ve antibiyotik direnç artışının önlenmesi açısından önem arz etmektedir. Hasta yatış süresinin uzamasının engellenmesi, ekonomik olarak kayıpların azaltılması ve pek çok yönden insan sağlığına katkı sağlaması için kolay bir yöntem olan multipleks PZR ile hızlı bir şekilde GSBL ve PMQR genlerinin belirlenmesi önerilmektedir.
Klebsiella pneumoniae (K. pneumoniae) are bacteria that produce dense sputum, causing inflammation and permanent damage to the lungs. In addition to pneumonia, they can cause infection, urinary tract infection (UTI), diarrhea, upper respiratory tract infection, wound infection, meningitis, bacteremia and septicemia at the surgical site. Sepsis or septic shock develops as a result of bacteria contaminating the blood. In this study, it was aimed to determine ESBL production characteristics and antibiotic susceptibilities of 192 K. pneumoniae strains isolated from clinical materials (55.2 % urine) in hospitals in Konya province both phenotypically and genotypically and to show the presence of plasmid mediated quinolone resistance (PMQR) genes. ESBL production characteristics were determined by phenotypic double disk synergy method. Antibiotic susceptibility tests were performed using the disk diffusion method recommended by Bauer-Kirby. For the antibiogram test and the double disc synergy method, bacterial solutions prepared at 0.5 Macfarland density were spread over the Mueller-Hinton agar surface and antibiotic discs were placed at certain distances. After the incubation, the zone diameters were measured and the resistant / susceptible strains were determined. In order to determine ESBL positives genotypically in K. pneumoniae strains, the presence of blaCTX-M, blaTEM, blaOXA, blaSHV beta-lactamase genes were screened by multiplex PCR method using specific primer pairs. Expressing plasmid mediated quinolone resistance in all K. pneumoniae isolates; qnrA (630 bp), qnrD (581 bp), qnrB (488 bp), qnrS (428 bp), oqxAB (313 bp), aac (6 ') - Ib-cr (260 bp), qepA (218 bp), qnrC (118 bp) genes were screened by multiplex PCR using specific primer pairs. 36 (18.75%) of 192 K. pneumoniae isolates were phenotypically and 186 (96.87%) were genotypically ESBL positive. According to the antibiogram results of K. pneumoniae strains, the highest resistance rate among 19 antibiotics tested was 97.9% against Ampicillin, while the lowest resistance antibiotic was Colistin (10mg) (11.45%). All of the strains were found to be resistant to at least one or more antibiotics and there were 113 different resistance profiles among the strains. According to the multiple antibiotic resistance (MAR) index, it was determined that 72% (139) of all K. pneumoniae strains had a resistance index higher than 0.2. This shows that the strains are isolated from regions where antibiotic use is high. According to the prevalence of ESBL genes in 192 K. pneumoniae strains, in 184 (95.83%) strains where they contain the most common blaSHV gene, blaTEM in 95 (49.47%) strains in the second place, blaOXA in 87 (45.31%) strains in the third place, and blaCTX-M gene in at least 27 (14.06%) strains. The highest rate of 26.5% was found to be the ESBL gene combination in which only blaSHV + blaTEM + blaOXA genes together. As a result of multiplex PCR of 8 PMQR genes of all K. pneumoniae isolates, it was determined that the most common gene was oqxAB gene found in 156 strains (81.25%). The least common gene was revealed to be the qnrC gene in 7 strains (3.6%). The highest rate of 21.87% was found to be the combination of only oqxAB + aac (6 ')-Ib-cr + qnrD, PMQR gene combination. This study demonstrated the importance of detecting ESBL-producing strains and PMQR genes for effective treatment. It is important in terms of preventing the unnecessary and wrong use of antibiotics and preventing the increase in antibiotic resistance, which is one of the major problems in the clinic. It is recommended to quickly determine ESBL and PMQR genes with multiplex PCR, which is an easy method to prevent prolongation of hospitalization, reduce losses economically and contribute to human health in many ways.
Klebsiella pneumoniae (K. pneumoniae) are bacteria that produce dense sputum, causing inflammation and permanent damage to the lungs. In addition to pneumonia, they can cause infection, urinary tract infection (UTI), diarrhea, upper respiratory tract infection, wound infection, meningitis, bacteremia and septicemia at the surgical site. Sepsis or septic shock develops as a result of bacteria contaminating the blood. In this study, it was aimed to determine ESBL production characteristics and antibiotic susceptibilities of 192 K. pneumoniae strains isolated from clinical materials (55.2 % urine) in hospitals in Konya province both phenotypically and genotypically and to show the presence of plasmid mediated quinolone resistance (PMQR) genes. ESBL production characteristics were determined by phenotypic double disk synergy method. Antibiotic susceptibility tests were performed using the disk diffusion method recommended by Bauer-Kirby. For the antibiogram test and the double disc synergy method, bacterial solutions prepared at 0.5 Macfarland density were spread over the Mueller-Hinton agar surface and antibiotic discs were placed at certain distances. After the incubation, the zone diameters were measured and the resistant / susceptible strains were determined. In order to determine ESBL positives genotypically in K. pneumoniae strains, the presence of blaCTX-M, blaTEM, blaOXA, blaSHV beta-lactamase genes were screened by multiplex PCR method using specific primer pairs. Expressing plasmid mediated quinolone resistance in all K. pneumoniae isolates; qnrA (630 bp), qnrD (581 bp), qnrB (488 bp), qnrS (428 bp), oqxAB (313 bp), aac (6 ') - Ib-cr (260 bp), qepA (218 bp), qnrC (118 bp) genes were screened by multiplex PCR using specific primer pairs. 36 (18.75%) of 192 K. pneumoniae isolates were phenotypically and 186 (96.87%) were genotypically ESBL positive. According to the antibiogram results of K. pneumoniae strains, the highest resistance rate among 19 antibiotics tested was 97.9% against Ampicillin, while the lowest resistance antibiotic was Colistin (10mg) (11.45%). All of the strains were found to be resistant to at least one or more antibiotics and there were 113 different resistance profiles among the strains. According to the multiple antibiotic resistance (MAR) index, it was determined that 72% (139) of all K. pneumoniae strains had a resistance index higher than 0.2. This shows that the strains are isolated from regions where antibiotic use is high. According to the prevalence of ESBL genes in 192 K. pneumoniae strains, in 184 (95.83%) strains where they contain the most common blaSHV gene, blaTEM in 95 (49.47%) strains in the second place, blaOXA in 87 (45.31%) strains in the third place, and blaCTX-M gene in at least 27 (14.06%) strains. The highest rate of 26.5% was found to be the ESBL gene combination in which only blaSHV + blaTEM + blaOXA genes together. As a result of multiplex PCR of 8 PMQR genes of all K. pneumoniae isolates, it was determined that the most common gene was oqxAB gene found in 156 strains (81.25%). The least common gene was revealed to be the qnrC gene in 7 strains (3.6%). The highest rate of 21.87% was found to be the combination of only oqxAB + aac (6 ')-Ib-cr + qnrD, PMQR gene combination. This study demonstrated the importance of detecting ESBL-producing strains and PMQR genes for effective treatment. It is important in terms of preventing the unnecessary and wrong use of antibiotics and preventing the increase in antibiotic resistance, which is one of the major problems in the clinic. It is recommended to quickly determine ESBL and PMQR genes with multiplex PCR, which is an easy method to prevent prolongation of hospitalization, reduce losses economically and contribute to human health in many ways.
Açıklama
Anahtar Kelimeler
K. pneumoniae, ÇAD indeksi, GSBL genleri, PMQR genleri, Multipleks PZR, MAR index, ESBL genes, PMQR genes, Multiplex PCR
Kaynak
WoS Q Değeri
Scopus Q Değeri
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Künye
Obalı, İ. (2020). Geniş spektrumlu beta laktamaz üreten ve üretmeyen klinik klebsiella pneumoniae suşlarında antibiyotik duyarlılıkları, blaCTX-M, blaTEM, blaOXA, blaSHV GSBL genlerinin ve PMQR genlerinin moleküler karakterizasyonu. (Doktora Tezi). Selçuk Üniversitesi, Fen Bilimleri Enstitüsü, Konya