Theses supervised by Prof. Dr. Füsun Can
10 theses · Koç University, Başkent University
Pseudomonas aeruginosa SST235 high-risk clone has unique adaptive response mechanisms to colistin stress
The dissemination of Pseudomonas aeruginosa (P.aeruginosa) ST235 high-risk clone is a public health threat. The emergence of antimicrobial resistance limits treatment options for P.aeruginosa infections. We aimed to identify new target sites for anti-virulent and antimicrobial therapy for multidrug-resistant ST235 P.aeruginosa infections by exploring metagenomic and transcriptomic responses of colistin-resistant P.aeruginosa. The study was performed in two stages. The twenty-seven patients diagnosed with colistin-resistant P.aeruginosa infection were included in the first resistant cohort. Secondly, two patients diagnosed with colistin-susceptible P.aeruginosa were included in the longitudinal study, and colistin tolerant/persister cells were induced under high colistin concentrations. The virulence genes and the genes related to persister formation were analyzed. Metagenomic analyses were performed to determine sequence type and the single nucleotide polymorphism (SNP). RNAseq was conducted to illuminate altered pathways. All assays were performed with Illumina HiseqXten/Novaseq sequencing platform. Outer membrane porin protein expression levels were studied with immunoblotting. The virulence scores of the isolates were determined by in vivo Caenorhabditis elegans (C. elegans) fertility model. We observed that the mortality rate was significantly higher in the cases infected with ST235 (81.8%) than in non-ST235 cases (36.4%) (p=0.0237). Molecular analysis showed that ST235 clones had amino acid substitutions in the pyoverdine biosynthesis genes of pvdA, pvdD, pvdJ, and pvdL, respectively. Besides, pvdD and pvdJ genes were downregulated in ST235 clones compared to NONST235 (p=0.0004). However, pyoverdine secretion was not significantly different in ST235 than the other clones (59.996 vs. 74.112 rfu/OD). In the longitudinal study, the metagenomic analysis presented that tolerant cell had genotypically the same missense mutations as susceptible and resistant isolates in persister-related genes. However, transcriptomic studies illuminated that tolerance cells expressed more DNA/repair/SOS response protein repair and stringent response genes than susceptible and resistant cells. Moreover, tolerance cells had the highest ((p)ppGpp synthesis gene relA expression compared to susceptible and resistant isolates. Colistin-resistant P.aeruginosa ST235 had completely different pyoverdine systems than other clones. These modifications in pyoverdine could contribute to the high virulence of the ST235 clone and should be considered as a target for anti-virulence therapy. The upregulation of the DNA repair/SOS response gene triggers the formation of colistin tolerant cells without alteration in genotype. Tolerome formation occurring in colistin susceptible isolates may play a significant role in the development of colistin resistance. Preventing the development of tolerance may offer a new strategy for delaying the emergence of resistance.
Effect of BNT162b2 and CoronaVac boosters on humoral immunity of individuals previously fully vaccinated with CoronaVac against SARS-CoV-2: A longitudinal study
It is essential to know about the immune response levels after booster doses of the two different types of vaccines, the mRNA and the inactivated, currently used against COVID-19. For this purpose, it was aimed to determine the effects of BNT162b2 (BNT) and CoronaVac (CV) boosters on the humoral immunity of individuals who had two doses of CV vaccination. For the estimation of humoral immune responses, live SARS-CoV-2 virus was isolated from nasopharyngeal swabs of SARS-CoV-2 PCR test positive patient and grown on the Vero E6 cells. After the confirmation of growth and estimation of the TCID50 for the virus, the virus was used in the PRNT50 experiments. For the study the healthcare workers who had no history of Covid-19 infection and were immunized with two doses of inactivated vaccine CoronaVac were included. Blood samples from these individuals were collected at different time points. From whole blood samples, PBMCs and sera were isolated. PBMCs were used for the flow cytometry experiments and sera were used for the neutralizing antibody experiments. In the neutralizing assay, the PRNT50 titer was calculated a dilution in which half of the virus at 10-3 PFU/ml concentration was inactivated and was selected for everyone in all time points. The percentage of B cell subtypes and proportion of memory B cells were estimated for each time point by staining with specific B cell markers (Anti-CD19, Anti-CD20, Anti-IgG, Anti- CD27, Anti- CD14, Anti-CD38 and Anti-CD56). After staining, samples were run by Attune flow cytometry. After the experiments, a 3.38-fold increase in neutralizing antibody geometric mean titers (Neutralizing Antibody, GM, 78.69) was found one month after BNT162b2 booster and maintained at the third month (Neutralizing antibody, GM, 80). Nevertheless, in the CoronaVac booster group, significantly lower neutralizing antibodies, GMT than BNT162b2 after 1 month and 3 months were observed (21.44 and 28.44, respectively) (p<0.001). In the B cell part, significantly higher expression of memory B cell and memory B cells expressing IgG was observed in the BNT162b2 booster group when compared with CoronaVac booster group both in 1st and 3rd month after booster vaccination. The ratio of effector memory B cells 1st month after vaccination in the CoronaVac booster group was significantly higher than BNT162b2 booster group (p=0.0263).
Prevalence and immune modulator role of the peg344 gene among hypervirulent Klebsiella pneumoniae
Hypervirulent Klebsiella pneumoniae (hvKp) is an emerging pathogen threatening healthy and immunocompromised patients. Since hvKp first appeared in Taiwan in 1986, its prevalence has increased drastically around the globe. Yet, there is no study focusing on hypervirulent strains in Turkey. peg344, iutA, and iroB are the highly accurate biomarker genes for hypervirulence. However, the pathogenesis mechanism of the peg344 putative metabolite transporter is still unclear. This study aims to elucidate the hypervirulence surveillance in Turkey and to understand the immune modulator role of the peg344 gene in vivo and in vitro infection models. In total, 230 K.pneumoniae isolates isolated from ten different healthcare centers in Turkey between January 2015 to March 2023 were included in the study. All strains were screened for hypervirulence genes peg344, iutA, and rmpA. The isolates harboring at least two of the hypervirulence gene were categorized as hypervirulence gene K.pneumoniae. The isolates which exhibited a hypermucoviscous phenotype in string test or virulent phenotype in 48-hour C.elegans fertility assay were classified as hypervirulent. The hypervirulence prevalence between 2022-2023 was significantly higher than 2017-2018 (p=0.024), and 2015-2016 (p<0.0001). The most prominent ST types were ST2096, and ST23 (30.77% and 23.08% respectively); K1 and K2 were the most common capsule serotypes among the hypervirulent cohort (46.15% and 15.38% respectively). THP-1-driven macrophage cells were infected with selected isolates including cKp, hgKp, hvKp, and Δpeg344-mutant isolates. hvKp isolates had decreased the viability of macrophages (from 91.5% to 81.5%) and increased the M2 convergence ratio at 2 hours of infection. Macrophages could inhibit the cKp isolates, but hvKp isolates were able to grow inside the macrophages at 3 hours of infection. Also, hvKp infection secreted less TNFα than cKp infection (368, 1462 pg/mL respectively). The peg344 deletion decreased the bacterial growth (from 430% to -3%) within the macrophages and recovered the TNFα secretion levels (from 203 to 824 pg/mL). In conclusion, hypervirulent K.pneumoniae is an emerging pathogen, and a serious concern in our region just like in other parts of the world. The peg344 gene is an accurate biomarker for differentiation in this region. The hypophagia of macrophages and the suppressed proinflammatory responses in the early stages contribute to the severe infections caused by hypervirulent clones. The peg344 gene plays an important role in bacteria's resistance against phagocytosis by macrophage cells.
Investigation of the relationship between vaginal dysbiosis with the activation of genital and intestinal inflammation during the pregnancy in an in vivo mouse model
The vaginal microbiota dysbiosis is represented by a low rate of Lactobacillus and several facultative pathogens such as Gardnerella, Atopobium, and Prevotella. A low abundance of Lactobacillus crispatus and a high abundance Gardnerella vaginalis and Atopobium vaginae species are significantly associated with preterm birth. The host-microbiota has been shown to regulate maternal and fetal immune interaction and birth outcomes. Bacterial taxa associated with dysbiosis have been found to stimulate proinflammatory cytokines that induce preterm labor. Progesterone treatment is one of the targeted therapies to prevent premature birth. The study aims to investigate the role of dysbiotic microbiota in the inflammatory process in a mouse model and also the effect of progesterone treatment on immune modulation in the genital and intestinal systems during pregnancy. Our mouse model revealed that vaginal exposure to dysbiotic microbiota showed an increasing trend of proinflammatory cytokine levels in the uterus and pathological inflammation through macrophage accumulation, resulting in 28% preterm birth. After the progesterone treatment in the dysbiosis group, TNFa and IL-6 levels were decreased. Besides, the macrophage density in the uterus was reduced, and less cellular damage in the placenta was observed. Analyzing the vaginal microbiota before or during pregnancy may support the decision for initiation of progesterone therapy. Besides, our results may guide new strategies like personalized treatments with novel drugs for preventing preterm birth.
Impact of colistin resistance on adaptive virulence mechanisms of Acinetobacter baumannii
Colistin resistance in Acinetobacter baumannii is an emerging problem that limits antimicrobial therapy options. In this study, we aimed to identify the adaptation of A. baumannii to antimicrobial and environmental stress by interchanging virulence traits. This study included two patient cohorts. The first cohort consists of 2 pairs of colistin-susceptible (K408, K1007) and colistin-resistant (K409, K1006) clonal isolates from 2 individual patients. The second cohort consists of 21 isolates (ColS=11, ColR=10) from 21 patients. Colistin susceptible isolates were exposed to in vitro colistin induction for 50 generations with and without exposure to HgCl2. The selected cell populations of colistin-susceptible, resistant, and in vitro-resistant induced isolates were subjected to DNA and RNA sequencing and phenotypic assays. Comparative virulence traits were examined using SNP analysis, transcriptomic analysis, biofilm formation assay, C. elegans fertility assay, bacterial adherence, cellular internalization, and cytokine expression assays. In the in vitro colistin induction assay, K408 gained colistin resistance on the corresponding day of clinical resistance (K408-G25) and got resensitized to colistin in the consecutive generation (K408-G26) while K1007 did not gain colistin resistance amongst 50 generations. On the other hand, in the in vitro colistin and HgCl2 co-exposure, K408 and K1007 developed resistance on 3rd generation and 5th generation, respectively. A significant upregulation of ompW, ata, and adeFGH genes on K408-G25 was followed by a downregulation upon resensitization to colistin (G26). Despite the upregulation of the ompW gene in transcriptomic analysis, the ompW protein disappeared on K408-G25 and recovered in the resensitized generation (G26). In parallel, disrupted cell membrane integrity and serum resistance activity were recovered in K408-G26. The ΔompW K408 strain exhibited significant downregulation of OMPs (ompA, carO) and adeFGH efflux pump operon (p=0.0026). Bacterial adherence and intercellular internalization assays revealed that colistin-resistant isolates (K408 G25, K408HG3, K409) exhibited lower affinity of adherence and invasion compared to the colistin susceptible cells. In parallel, proinflammatory cytokine expression was significantly higher in colistin susceptible cells (p= 0.0395). In the 2nd cohort, ompW was significantly downregulated with lower protein abundance (p=0.004). Additionally, in the resistant cohort, the pmrCAB and adeFGH operons were significantly upregulated (p<0.001). Eventually, clinically colistin resistant and in vitro colistin resistance induced isolates (K408 G25, K408 HG3, K409, K1006) exhibited lower C. elegans virulence scores compared to the colistin susceptible (K408, K1007) and colistin resensitized (K408 G26) cells. These results were further confirmed by the second cohort with statistically lower virulence scores (p=0.0224). In this study, we showed that, during the early stages of response to colistin stress until the development of chromosomal resistance, Acinetobacter baumannii regulates adhesion, efflux pump, and serum resistance associated virulence traits. Moreover, mercury exposure plays an important role in facilitating colistin resistance. Colistin resistant cells lose their ability to cell invasion and intercellular persistence. Eventually, the ompW protein is an unstable outer membrane protein which may result in suboptimal outcomes for the ompW targeted immunisation strategies in the prevention of colistin resistant A. baumannii infections.
Hipervirülan Klebsiella pneumoniae'da peg-344'ün makrofajlarin otofaji yanitlarini baskilamadaki rolü
Hypervirulent Klebsiella pneumoniae (hvKp) poses a significant challenge due to its increased pathogenicity and increasing antibiotic resistance. Because of this reason it is important to understand the pathogenesis of the bacteria as well as the alternative therapies. In this study we aimed to address the role of the peg-344 gene of Klebsiella pneumoniae in autophagy and immune response suppression. Intracellular survival revealed that hvKp is significantly less internalized by macrophages than cKp (p=0.0001). hvKp showed 3-log higher intracellular CFU compared to cKp at 24H (p=0.0022) while causing lower cytotoxicity on macrophages. When autophagy is externally activated with Torin 1, the intracellular growth of hvKp still persists. Infection with hvKp significantly increases autophagy activation (Chloroquine, conversion of LC3-I to LC3-II). However, the persistent presence of p62 and low cytotoxicity suggest the bacterium blocks the fusion of phagosomes with lysosomes, enabling its survival within macrophages. ELISA experiments showed significantly lower TNF-α and IL-1β release against hvKp isolates was observed, showing that hvKp escapes macrophage immune responses. Deletion of peg-344 gene reversed the lower internalization and intracellular survival compared to peg-344 intact isolate. Peg-344 deletion also caused the loss of lower pro-inflammatory cytokine release and autophagy activation. All of these findings further support the idea that peg-344 may suppress macrophage autophagy responses. In conclusion, the study suggests that hypervirulent Klebsiella employs mechanisms to survive within macrophages, evade immune responses, and suppress autophagy. The peg-344 gene appears to play a significant role in these processes, contributing to both intracellular survival and immune/autophagy evasion.
Kateter ilişkili hastane enfeksiyonlarında sık karşılaşılan biyofilm oluşturan bakterilerde, biyofilm ilişkili antibiyotik direncinin araştırılması
Hastaneye yatıs sürecinde tanı ve tedavi amaçlı kullanılan yabancı cisimlerden kateterlerin, en önemli komplikasyonları arasında lokal ve sistemik enfeksiyon riski yer almaktadır. Bu çalısma kateter yüzeylerinde mikroorganizmalar tarafından olusturulan biyofilm yapısının tedaviye direnci artıran ve tedavi sürecini zorlastıran önemli bir etken olduğuna dayanılarak yapılmıstır. Çalısmada Baskent Üniversitesi Tıp Fakültesi Ankara Hastanesi'nde 2006-2009 yılları arasında çesitli servislerde ve yoğun bakımlarda yatarak tedavi gören hastaların kateterlerinde üremis metisilin dirençli S.aureus (MRSA), metisilin dirençli koagülaz negatif stafilokok (MRKNS) ve P.aeruginosa izolatlarının planktonik hücre asamasında ve in vitro kosullarda biyofilm yaptırıldıktan sonra sesil hücrelerinin çesitli antibiyotiklere duyarlılıkları arastırılmıstır. Antibiyotiklerin seçiminde MRSA ve MRKNS susları için vankomisin, siprofloksasin, rifampisin, gentamisin, meropenem, tigesiklin, linezolid, seftazidim ve sefazolin tercih edilirken, P.aeruginosa susları için de siprofloksasin, gentamisin, meropenem, tigesiklin, piperasilin, seftazidim ve amikasin çalısmaya alınmıstır. Seçilen bu antibiyotikler ile önce planktonik hücrelerde duyarlılık arastırılmıs, böylece minimal inhibitör konsantrasyonları (MĐK) ve minimal bakterisidal konsantrasyonları (MBK) belirlenmistir. Daha sonra her bir sus ile biyofilm olusturulup sesil hücrelerde aynı antibiyotikler ile yine MĐK ve MBK değerleri belirlenmistir. Ardından ikili ve üçlü antibiyotik kombinasyonları olusturulup, çoklu kombine bakterisidal test (ÇKBT) yöntemi ile hem planktonik hem de biyofilmde etkinlikleri belirlenmistir. Her bir antibiyotik ile planktonik ve biyofilm hücrelerinde elde edilen veri seti istatistiksel olarak iki oran z testi ile analiz edilmistir. Sonuç olarak; sesil hücrelerde planktonik forma göre genel olarak duyarlılıklarda anlamlı azalmalar tespit edilmistir. Hem ikili hem de üçlü kombinasyonlar ile yapılan çalısmaların sonucunda da sesil hücrelerin kombinasyonların çoğuna duyarlılıklarında anlamlı azalma olduğu görülmüstür. Planktonik ve sesil hücrelere kombinasyonların etkinliğinin karsılastırılmasında, susların % 90'ından fazlasını inhibe etme açısından, ikili kombinasyonlar ile üçlüler arasında anlamlı bir fark tespit edilmemistir.
Acinetobacter baumannii`nin kolistin maruziyetine uyumu: İn vivo ve in vitro deneyimler
Acinetobacter baumannii (A.baumannii) is one of the emerging pathogens which causes severe infections with high mortality. Multidrug-resistant (MDR) and colistin-resistant A.baumannii spread worldwide especially in healthcare centers. A.baumannii has two genetic mechanisms of colistin resistance: Complete loss of LPS as a result of mutations in lpx genes and point mutations in pmrCAB operon. Both mechanisms cause structural changes and eventually prevent colistin binding to cell wall. However, triggering factors and evolutionary mechanisms of colistin resistance are still not clearly understood. In this study, we aimed to mimic induction of colistin resistance by colistin exposure, follow-up the progression of resistance and compare in vitro results with in vivo resistance identified in an isolate from a patient. A 35-years old female patient admitted in VKV American Hospital (Istanbul) intensive care unit between February and March 2016 was chosen for the study. A total of four colistin susceptible and one colistin resistant A.baumannii isolates were collected. Patient's clinical data and outcome were recorded. Daily serial passages of susceptible isolates in presence of colistin at 1 mg/L concentration were performed to mimic development of colistin resistance in vitro by colistin exposure. Minimum inhibitory concentrations (MICs) were measured by broth microdilution. The colistin-resistant isolate and certain generations of in vitro experiment were chosen for molecular analyses. The pmrCAB complete operon and lpxA, lpxC, and lpxD genes were sequenced. The expression levels of pmrC, pmrA, and pmrB were studied by qRT-PCR. The patient's data and the results of molecular tests were compared. The pmrA, pmrB and pmrC genes were 1.6, 1.74 and 1.72 times overexpressed in colistin resistant clinical isolate than susceptible one. Colistin resistance was identified in the patient at 25th day of the colistin therapy. In 38 serial passages of four susceptible isolates, colistin MIC values were above breakpoint level of 2 mg/L after first passage. At the 26th generation of experiment, pmrC expressions of all isolates reached to a peak level (2.11; 29.65-fold). Moreover, in 2 of the four isolates pmrA (1.97 and 8.54-fold) and pmrB (2.31 and 11.24-fold) expressions were at the highest level in the same generation. We also detected multiple insertions in pmrA, pmrB, pmrC, lpxA, lpxC and lpxD genes. In conclusion, upregulation of pmrC, pmrA, and pmrB in combination with insertions in these genes and lpx genes may trigger development of colistin resistance. The highest pmrCAB expressions after 26th day of exposure and isolation of resistant strain from the patient at 25th day of colistin therapy suggested us that long term therapy is required for development of colistin resistance in A.baumannii. Therefore, duration of colistin use and combined therapy options should be considered and controlled properly during colistin therapy.
E. coli ST131 enfeksiyonlarının tedavisinde yeni bir yaklaşım: Nano taşıyıcı ile hedefe yönlendirilmiş antimikrobiyal ajanlar
Escherichia coli (E.coli) ST131 is a globally disseminating high risk clone associated with multidrug resistance and failure of antimicrobial therapy. There is an urgent need for alternative approaches to treat E.coli ST131 infections. Our aim was to design a SPION (Super Paramagnetic Iron Oxide Nanoparticle) based drug delivery system targeting E.coli ST131 clone. In this study, we studied on a quinolone resistant uropathogenic E.coli ST131 isolate to assess the efficacy of targeted delivery system. Polyacrylic acid (PAA) coated SPIONs were synthesized by dissolving FeCl2 and FeCl3 salts in deoxygenated water and coating SPIONs with PAA. Nanoparticle size was 33.2 nm. After characterization of the particles regarding hydrodynamic and crystal size, zeta potential for surface charge and stability, SPIONs were conjugated with mannoside analog (4-aminophenyl α-D-mannopyranoside) to target fimH adhesin of E.coli ST131. Subsequently, ciprofloxacin was attached to the SPION-Mannoside complex. Specific interaction and antibacterial activity of delivery system were examined on planktonic and sessile cells of E.coli ST131 isolate by using growth inhibition, confocal imaging, and scanning electron microscopy (SEM) techniques. Furthermore, photothermal therapy (PTT) was applied at power of 1150 mW for ten minutes in combination with SPION exposure. SPIONs at 25 μg/ml concentration (alone or conjugated with mannoside) did not cause significant growth inhibition both on planktonic and sessile cells of E.coli ST131. The mean colony count of E.coli ST131 planktonic cells after incubation with SPION and SPION-Mannoside were 2.26x1012 and 9.89x1011 CFU/ml, respectively while it was 1.22x1012 CFU/ml in control culture. In biofilm, incubation with mannoside conjugated 25 μg/ml SPION resulted in 1.55x1010 CFU/ml mean colony count while SPION and control cultures revealed 2.04x1010 CFU/ml and 7.76x109 CFU/ml bacterial growth, respectively. Confocal microscopy indicated more specific and enhanced attachment of SPION-Mannoside particles to the surface of E.coli ST131 compared to SPION alone. However, the SPION particles were seen accumulated on biofilm surface and did not penetrate inside the biofilm matrix. After conjugation of ciprofloxacin to SPION-mannoside, the delivery system did not cause a significant reduction in growth of planktonic and sessile cells (log change in mean CFU/ml lower than 2-log). The mean colony counts of planktonic cells were 1.70x1010 and 4.32x1012 CFU/ml at ciprofloxacin-mannoside conjugated SPION and control cultures, respectively. In biofilm, incubation with ciprofloxacin-mannoside conjugated SPION resulted in growth of 1.66x1010 CFU/ml mean colony count while it was 3.24x1010 CFU/ml for control. PTT application did not enhance the efficiency of delivery system. No significant decrease in bacterial growth was observed. SPION based targeted therapy is a unique and promising approach to treat persistent E.coli ST131 infections. Our future goals are to integrate quinolone derivatives and efflux pump inhibitors to our mannoside conjugated SPION cargo system.
Behçet üveitinin immünopatogenezi ve IFN-α-2A tedavisine karşı immünyanıt
This study was conducted to investigate a) The immunopathogenesis of BD and BD Uveitis and b) the effect of interferon alfa-2a, on adaptive and innate immunity in patients with Behçet uveitis. For the adaptive immune responses, peripheral blood regulatory T Cells and TH17 cells were analyzed. For the innate immune responses, mRNA expression of TLRs 2, 3, 4, 8 and 9 on monocytes were analyzed. Nineteen patients with active Behçet uveitis who were refractory to conventional immunomodulatory therapy and were administered interferon alfa-2a; and age- and sex-matched healthy control subjects were consecutively enrolled from August 2016 to September 2017. Peripheral blood from patients was collected before treatment and after angiographic remission was achieved with interferon alfa-2a. Treg and TH17 percentages were determined as CD3+CD4+FoxP3+ and CD3+CD4+IL17A+ (after in vitro stimulation) respectively by flow-cytometry. TH17 RORɣT mRNA expression was verified by real-time PCR (RT PCR). Cytokines released from Treg and TH17 cells were detected by ELISA in culture supernatants. RT PCR was used to measure mRNA levels of TLRs 2, 3, 4, 8 and 9 from monocytes. R Statistical Software v.3.5.1 was used for statistics and Mann-Whitney U and Wilcoxon tests were used for analyses. Treg and TH17 percentages and TH17 RORɣT mRNA expression were found significantly elevated in BU patients. Patients had significantly lower Treg IL-10 concentration than controls. IL-17A, IL-6, IL-21, IL-22, IL-23, IFN-ɣ, and TNF-α concentrations from Th17 Cells were found to be significantly higher in patients than in controls. All TLR expressions were found significantly elevated in monocytes of BU patients. Interferon alfa-2a (median total dose: 330 million international units, median duration: 5 months) led to remission in BU patients. A significant reversal of Treg and TH17 percentage, TH17 RORɣT mRNA expression, Treg and TH17 cytokine release, and TLR expressions from monocytes were observed in BU patients after interferon therapy. Despite a paradoxical increase in Treg population, impaired IL-10 production suggests Treg dysfunction in patients with active BU. Interferon alfa-2a restored Treg functions, induces IL-10 production from Tregs, suppresses TH17s and downregulates TLR expression in monocytes.