Hastene Kaynaklı Klebsiella pneumoniae’ya Yönelik İnaktif ve Rekombinant Aşı Denemeleri
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Dosyalar
Tarih
2022
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Selçuk Üniversitesi Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Hastane kaynaklı enfeksiyonlarda sıklıkla etken olarak izole edilen K. pneumoniae
günümüzde toplumsal kökenli enfeksiyonlarda da karşımıza çıkmaktadır. Özellikle çoklu ilaca
dirençli suşlarla oluşan enfeksiyonlarda tedavide yaşanan problemler ölüm oranlarının artmasına
neden olmaktadır. Giderek artan direnç düzeyleri tedavide altın standart olarak görülen
antibiyotiklerin ne derece yetersiz kaldığını göstermektedir. Tedavideki sıkıntılara bağlı artan ölüm
oranları immünoterapi ve aşı gibi koruyucu protokolleri ön plana çıkarmaktadır. Bu çalışmada 2019
yılında Konya Numune Hastanesi Yoğun Bakım Ünitesinde tedavi gören hastalardan izole edilen K.
pneumoniae izolatlarının fenotipik ve gonotipik özelliklerinin araştırılması, artan antibiyotik direnç
düzeylerinin belirlenmesi, yaygın kapsüller tipin belirlenmesi, hipervirulant izolatların tespiti,
rekombinant rmpA proteininin elde edilmesi, uygun aşı formülasyonlarının hazırlanarak
etkinliklerinin seropotens ve çelınç fare gruplarında değerlendirilmesi amaçlanmıştır.
Bu çalışmada, Konya Numune Hastanesi yoğun bakım ünitelerinde 2019 yılında tedavi
gören hastalardan izole edilen K. pneumoniae izolatları kullanıldı. Bütün izolatların antibiyotik
dirençlilikleri EUCAST sisteminin önerdiği kriterlere uygun olarak yapıldı. İzolatların karbapenamaz
ve β-laktamaz enzim aktiviteleri klasik (MBL, MHT ve çift disk sinerjisma) ve moleküler tekniklerle
değerlendirildi. İzolatların virulans, kapsüller ve direnç ile ilişkili gen bölgeleri PZR ile gösterildi.
Biyofilm oluşturma yetenekleri kalitatif ve kantitatif olarak ölçüldü. İzotatların akrabalıkları RAPD PZR ile gösterildi. Hipervirulant izolatlar string test, koloni morfolojisi ve rmpA gen varlığının PZR
ile gösterilmesi ile tespit edildi. Klasik ve moleküler işlemler sonucunda en uygun aşı suşları
belirlendi. Rekombinant olarak rmpA proteinini elde etmek için PZR ile amplifiye edilen gen pET
SUMO ekspresyon vektöre klonlanarak E. coli BL21(DE3)’e transforme edildi. İşlemin doğrulanması
için string test, koloni morfolojisi, sekans analizi ve PZR teknikleri kullanıldı. Rekombinanat olarak
üretilen proteinin değerlendirilmesinde SDS-PAGE ve Western Blot testleri kullanıldı. Hazırlanan
antijenler MontanideTM IMS 3012 VG, Complete Freund’s Adjuvant ve Aluminyum Hidroksit Jel
(Al(OH)3) ile kombine edildi ve yedi farklı aday aşı elde edildi. Sonuçlar hali hazırda K. pneumoniae
için ticari bir aşı bulunmaması nedeni ile Al(OH)3 ile hazırlanan inaktif aşı formülasyonu, negatif
kontrol ve hiperimmunizasyon ile elde edilen pozitif kontrol gruplarına ait sonuçlar ile karşılaştırıldı.
Hazırlanan aşıların etkinliklerinin karşılaştırmasında çelınç ve ELISA teknikleri kullanıldı.
Antibiyotik sınıflandırmalarına göre kaynaklar değerlendirildiğinde bronş ve yara kaynaklı
izolatların ExDR ve PDR oranlarının diğer izolatlara göre daha yüksek olduğu belirlendi. Bu direncin
oluşmasında GSBL, MHT ve MBL enzim aktivitelerinin (p<0,001) önemli rol oynadığı görüldü.
Enzim aktiviteleri ile ilişkili gen bölgeleri değerlendirildiğinde, CTXM, OXA, IMP, NDM ve KPC
varlığında (p=0,01) direncin pozitif yönde arttığı, kapsüller tiplendirmelerde ise K1, K54 ve K57’nin
direnç düzeylerinin (p<0.001) daha yüksek olduğu ve virulans gen bölgelerinden entB dışında diğer
genlerin tamamında (p<0,001) direncin arttığı görüldü. Bütün izolatlar arasında GSBL pozitifliği %
44,44 olarak belirlendi. GSBL üreten izolatlarda ki aminoglikozid direnç düzeyine bakıldığında
amikasinde % 50, gentamisinde % 56,33 direnç oluştuğu gözlemlendi. Kolistin direnç tespitinde
EUCAST’ta göre altın standart kabul edilen MIC yöntemine göre diğer sonuçlar karşılaştırıldığında
VITEC II sonuçlarının (p=0,11) tutarlı olmadığı; agar dilisyon yönteminin ise (p<0,001) kullanılabilir
bir yöntem olduğu belirlendi. Numunelerdeki Metallo β-Laktamaz enzim varlığının gösterilmesi her
üç karbapenem grubu antibiyotiklerdeki direnci göstermek için (p<0,001) güvenilir ve ucuz bir
xviii
yöntem olarak belirlendi. Strig test sonuçları rmpA PZR sonuçları ile karşılaştırıldığında her iki
yöntem arasındaki ilişkinin (p<0,001) anlamlı olduğu görüldü. Fenotipik ve genotipik uyuma göre
blaKPC, blaOXA, blaNDM, blaVIM, blaIMP genleri değerlendirildiğinde; karbapenem grubu antibiyotikler
arasında değişen düzeylerde ilişki olduğu, en anlamlı gen bölgelerinin blaOXA ve blaIMP (p<0,001)
olduğu bu nedenle karbapenamazların üretilmesinde bu bölgelerin sorumlu olabileceği sonucuna
varıldı. Aminoglikozit ile ilişkili gen bölgeleri VITEK II ve disk difüzyon sonuçları ile karşılaştırıldı.
Her iki antibiyotik için de VİTEC II sonuçlarının tutarlı olmadığı (p=0,35) ancak disk difüzyon
sunuçlarının uyumlu olduğu (p=0,02) görüldü. Genotiplendirme sonuçlarında Grup-B’de yer alan üç
izolatında β-laktamaz ve karbapenamaz üretmeyen, kolistin direnci göstermeyen ve biyofilm
oluşturmayan idrar kaynaklı numuneler olduğu, Grup-A’nın ise 7 farklı daldan meydana geldiği A-I
grubunda idrardan (% 72), A-II grubunda yaradan (% 64,3) izole idilenlerin yer aldığı; bronş
numunelerinin ise (% 46) A-I ve (% 42,4) A-II gruplarında eşit dağılım gösterdikleri görüldü. Aşı
suşları seçilirken çalışmaya dahil edilen izolatların % 94,44’ünde yaygın olarak tespit edilen K5 tipi
ve rekombinant aşı için HvKP izolatlarında HM fenotipin ortaya çıkmasından sorumlu gen bölgesi
olan rmpA rekombinant protein üretimi için seçilmiştir. Seropotens grubunda bütün aşı gruplarında
negatif kontrol grubu ile (p<0,001) yapılan karşılaştırmalarda sonuçların anlamlı olduğu ve pozitif
kontrol gruplarından daha yüksek antikor seviyelerine ulaşıldığı görülmektedir. Rekombinant protein
ile hazırlanan aşı gruplarında Odds oranının yüksek olması HvKP enfeksiyonlarında bu iki grubun
hayatta kalımı artırdığı yönünde yorumlanmıştır. Bütün bu veriler ışığında K. pneumoniae
enfeksiyonlarından korunmada ve/veya oluşan enfeksiyonlardaki dirençli ve HvKP suşlarının
yayılımının önlenmesinde aşı uygulamaları ön plana çıkmaktadır.
Sonuç olarak; bütün izolatlarda hızla yayılım gösteren antibiyotik direnç seviyeleri ve hızla
artan HvKP suşları nedeni ile tedavide yetersiz kalındığı görülmektedir. Bu çalışmada üç farklı
adjuvan ile oluşturulan yedi farklı aşı grubunda oluşan antikor seviyeleri negatif kontrol grubu ile
kıyaslandığında ve çelınç sonuçları ile değerlendirildiğinde bütün aşı gruplarının ölüm oranlarını
azalttığı oluşan antikor seviyelerinin hayatta kalımı arttırdığı görülmektedir.
K. pneumoniae, which is often isolated as a causative agent in hospital-acquired infections, is also encountered in community-acquired infections nowadays. Especially in infections caused by multi-drug resistant strains, problems in treatment cause an increase in mortality rates. The increasing resistance levels show the inadequacy of antibiotics, which are considered the gold standard for the treatment. The increasing mortality rates due to problems in the treatment highlight preventive protocols such as immunotherapy and vaccines. The aim of this study was to investigation of the phenotypic and gonotypic characteristics of K. pneumoniae isolates isolated from patients treated in the Intensive Care Unit of Konya Numune Hospital in 2019, determination of increasing antibiotic resistance levels, determination of common capsule type, detection of hypervirulent isolates, obtaining recombinant rmpA protein, preparation of appropriate vaccine formulations and evaluate their efficacy in seropotens and celinc mouse groups. In this study, K. pneumoniae isolates from patients treated in intensive care units of the Konya Numune Hospital in 2019 were used. The antibiotic resistance of all the isolates was determined in accordance with the criteria suggested by the EUCAST system. Carbapenemase and β lactamase enzyme activities of the isolates were evaluated by classical (MBL, MHT and double-disc synergism) and molecular techniques. The gene regions associated with virulence, capsules and resistance of the isolates were demonstrated by PCR. Biofilm forming abilities were measured qualitatively and quantitatively. The relatedness of isolates was demonstrated by RAPD-PCR. Hypervirulent isolates were detected by string test, colony morphology and PCR detection of the presence of the rmpA gene. As a result of the classical and molecular procedures, the most suitable vaccine strains were determined. In order to obtain the recombinant rmpA protein, the gene amplified by PCR was cloned into the pET SUMO expression vector and transformed into E. coli BL21(DE3). In order to verify the process, string testing, colony morphology, sequence analysis and PCR techniques were used. SDS-PAGE and Western Blot tests were used to evaluate the protein produced as recombinant. Prepared antigens were combined with MontanideTM IMS 3012 VG, Complete Freund's Adjuvant and Aluminum Hydroxide Gel (Al(OH)3) and seven different candidate vaccines were obtained. Since, so far, there is no commercial vaccine for K. Pneumoniae, the inactivated vaccine formulation prepared with Al(OH)3 was compared with the results of the negative control and the positive control groups obtained by hyperimmunization. Challenge and ELISA techniques were used to compare the efficacy of the prepared vaccines. When the sources were evaluated according to the antibiotic classifications, it was determined that the ExDR and PDR ratios of bronchial and wound-derived isolates were higher than in the other isolates. It was observed that ESBL, MHT and MBL enzyme activities (p<0.001) played an important role in forming this resistance. When the gene regions associated with enzyme activities were evaluated, it was found that the resistance increased positively in the presence of CTXM, OXA, IMP, NDM and KPC (p=0.01), whereas, in capsule typings, the resistance levels of K1, K54 and K57 were higher (p<0.001) being observed that, of the virulence gene regions, the resistance was increased in all genes (p<0.001) except entB. ESBL positivity was determined as 44.44% among all the isolates. When the aminoglycoside resistance level in ESBL producing isolates was examined, it was observed that resistance occurred in amikacin at 50% and in gentamicin at 56.33%. In the determination of colistin resistance, when the other results were compared according to the MIC method, which is xx accepted as the gold standard by the EUCAST, it was revealed that the results of VITEC II (p=0.11) were not consistent and that agar dilution method (p<0.001) was an applicable method. The presence of the Metallo β-Lactamase enzyme in the samples was determined as a reliable and inexpensive method to show the resistance in all three carbapenem group antibiotics (p<0.001). When the string test results were compared with the rmpA PCR results, the relationship (p<0.001) between both methods was found to be significant. When evaluating blaKPC, blaOXA, blaNDM, blaVIM, blaIMP genes according to phenotypic and genotypic compatibility; it was concluded that there was a relationship at varying levels between carbapenem group antibiotics, being the most significant gene regions blaOXA and blaIMP (p<0.001), therefore these regions might be responsible for the production of carbapenemases. The gene regions associated with aminoglycosides were compared with VITEK II and disc diffusion results. It was observed that VITEC II results were not consistent (p=0.35) for both antibiotics, but the disc diffusion results were coherent (p=0.02). In the genotyping results, it was observed that three isolates from urine-derived samples in the Group-B did not produce β-lactamases nor carbapenemases, did not show colistin resistance and did not form a biofilm; it was also noticed that the Group-A consisted of 7 different branches, including the isolates from urine (72%) in the A-I group, and those wound-isolated (64.3%) in the A-II group; while the bronchial samples showed an equal distribution in groups A-I (46%) and A-II (42.4%). While selecting the vaccine strains, it was found that 94,44% of the isolates included in the study were identified as K5 type, and as the rmpA is the gene region responsible for the emergence of H.M. phenotype in HvKP isolates, it was selected for the production of the recombinant protein. In the seropotency group, the comparison of all the vaccine groups with the negative control group (p<0.001) revealed that the results are significant and that higher antibody levels than in the positive control groups are reached. The high Odds ratio in the groups inoculated with a vaccine prepared with recombinant protein was interpreted as an increased survival rate in HvKP infections in these two groups. Considering all these data, vaccine applications come to the forefront in the prevention of K. pneumoniae infections and/or the prevention of the spread of resistant and HvKP strains in infections. As a result, it is observed that the treatment is insufficient due to the quickly spreading antibiotic resistance levels in all the isolates and rapidly increasing HvKP strains. In this study, when the antibody levels in seven different vaccine groups created with three different adjuvants are compared with the negative control group and evaluated with the results of the challenge, it is seen that the antibody levels in all the vaccine groups decrease the death rates and increase the survival rates.
K. pneumoniae, which is often isolated as a causative agent in hospital-acquired infections, is also encountered in community-acquired infections nowadays. Especially in infections caused by multi-drug resistant strains, problems in treatment cause an increase in mortality rates. The increasing resistance levels show the inadequacy of antibiotics, which are considered the gold standard for the treatment. The increasing mortality rates due to problems in the treatment highlight preventive protocols such as immunotherapy and vaccines. The aim of this study was to investigation of the phenotypic and gonotypic characteristics of K. pneumoniae isolates isolated from patients treated in the Intensive Care Unit of Konya Numune Hospital in 2019, determination of increasing antibiotic resistance levels, determination of common capsule type, detection of hypervirulent isolates, obtaining recombinant rmpA protein, preparation of appropriate vaccine formulations and evaluate their efficacy in seropotens and celinc mouse groups. In this study, K. pneumoniae isolates from patients treated in intensive care units of the Konya Numune Hospital in 2019 were used. The antibiotic resistance of all the isolates was determined in accordance with the criteria suggested by the EUCAST system. Carbapenemase and β lactamase enzyme activities of the isolates were evaluated by classical (MBL, MHT and double-disc synergism) and molecular techniques. The gene regions associated with virulence, capsules and resistance of the isolates were demonstrated by PCR. Biofilm forming abilities were measured qualitatively and quantitatively. The relatedness of isolates was demonstrated by RAPD-PCR. Hypervirulent isolates were detected by string test, colony morphology and PCR detection of the presence of the rmpA gene. As a result of the classical and molecular procedures, the most suitable vaccine strains were determined. In order to obtain the recombinant rmpA protein, the gene amplified by PCR was cloned into the pET SUMO expression vector and transformed into E. coli BL21(DE3). In order to verify the process, string testing, colony morphology, sequence analysis and PCR techniques were used. SDS-PAGE and Western Blot tests were used to evaluate the protein produced as recombinant. Prepared antigens were combined with MontanideTM IMS 3012 VG, Complete Freund's Adjuvant and Aluminum Hydroxide Gel (Al(OH)3) and seven different candidate vaccines were obtained. Since, so far, there is no commercial vaccine for K. Pneumoniae, the inactivated vaccine formulation prepared with Al(OH)3 was compared with the results of the negative control and the positive control groups obtained by hyperimmunization. Challenge and ELISA techniques were used to compare the efficacy of the prepared vaccines. When the sources were evaluated according to the antibiotic classifications, it was determined that the ExDR and PDR ratios of bronchial and wound-derived isolates were higher than in the other isolates. It was observed that ESBL, MHT and MBL enzyme activities (p<0.001) played an important role in forming this resistance. When the gene regions associated with enzyme activities were evaluated, it was found that the resistance increased positively in the presence of CTXM, OXA, IMP, NDM and KPC (p=0.01), whereas, in capsule typings, the resistance levels of K1, K54 and K57 were higher (p<0.001) being observed that, of the virulence gene regions, the resistance was increased in all genes (p<0.001) except entB. ESBL positivity was determined as 44.44% among all the isolates. When the aminoglycoside resistance level in ESBL producing isolates was examined, it was observed that resistance occurred in amikacin at 50% and in gentamicin at 56.33%. In the determination of colistin resistance, when the other results were compared according to the MIC method, which is xx accepted as the gold standard by the EUCAST, it was revealed that the results of VITEC II (p=0.11) were not consistent and that agar dilution method (p<0.001) was an applicable method. The presence of the Metallo β-Lactamase enzyme in the samples was determined as a reliable and inexpensive method to show the resistance in all three carbapenem group antibiotics (p<0.001). When the string test results were compared with the rmpA PCR results, the relationship (p<0.001) between both methods was found to be significant. When evaluating blaKPC, blaOXA, blaNDM, blaVIM, blaIMP genes according to phenotypic and genotypic compatibility; it was concluded that there was a relationship at varying levels between carbapenem group antibiotics, being the most significant gene regions blaOXA and blaIMP (p<0.001), therefore these regions might be responsible for the production of carbapenemases. The gene regions associated with aminoglycosides were compared with VITEK II and disc diffusion results. It was observed that VITEC II results were not consistent (p=0.35) for both antibiotics, but the disc diffusion results were coherent (p=0.02). In the genotyping results, it was observed that three isolates from urine-derived samples in the Group-B did not produce β-lactamases nor carbapenemases, did not show colistin resistance and did not form a biofilm; it was also noticed that the Group-A consisted of 7 different branches, including the isolates from urine (72%) in the A-I group, and those wound-isolated (64.3%) in the A-II group; while the bronchial samples showed an equal distribution in groups A-I (46%) and A-II (42.4%). While selecting the vaccine strains, it was found that 94,44% of the isolates included in the study were identified as K5 type, and as the rmpA is the gene region responsible for the emergence of H.M. phenotype in HvKP isolates, it was selected for the production of the recombinant protein. In the seropotency group, the comparison of all the vaccine groups with the negative control group (p<0.001) revealed that the results are significant and that higher antibody levels than in the positive control groups are reached. The high Odds ratio in the groups inoculated with a vaccine prepared with recombinant protein was interpreted as an increased survival rate in HvKP infections in these two groups. Considering all these data, vaccine applications come to the forefront in the prevention of K. pneumoniae infections and/or the prevention of the spread of resistant and HvKP strains in infections. As a result, it is observed that the treatment is insufficient due to the quickly spreading antibiotic resistance levels in all the isolates and rapidly increasing HvKP strains. In this study, when the antibody levels in seven different vaccine groups created with three different adjuvants are compared with the negative control group and evaluated with the results of the challenge, it is seen that the antibody levels in all the vaccine groups decrease the death rates and increase the survival rates.
Açıklama
Anahtar Kelimeler
K. pneumoniae, aşı, rmpA, direnç, vaccine, resistance
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
İlban, A., (2022). Hastene Kaynaklı Klebsiella pneumoniae’ya Yönelik İnaktif ve Rekombinant Aşı Denemeleri. (Doktora Tezi). Selçuk Üniversitesi, Sağlık Bilimleri Enstitüsü, Konya.
