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Öğe Determination of in vitro biofilm formation abilities of food borne salmonella enterica isolates(Trakya Üniversitesi Fen Bilimleri Enstitüsü, 2019) Aksoy, DenizSalmonellosis caused by non-typhodial Salmonella enterica serotypes is one of the most important food-borne diseases worldwide and biofilm structure formed by these pathogens provide a reservoir for food contamination and a source for infections. This study was performed in order to determine biofilm formation abilities of food borne Salmonella isolates on polystyrene and on air liquid interphase and their colony morphologies when grown on Congo Red Agar plates. 32 food-borne Salmonella strains isolated from retail chicken carcasses in Edirne province of Turkey and belonging to the Infantis, Enteritidis, Kentucky and Telaviv serotypes were used. The microtiter plate technique was used to determine biofilm formation abilities of the isolates on polystyrene surfaces by measuring the optical density (OD) values of the stained bacterial biofilms. The results showed that the strongest biofilm formation capacities of the isolates were observed at 22°C for 3 days of incubation. Although all isolates formed pellicle on the liquid-air interface at 22°C, only 13% of the isolates belonging to the Infantis, Kentucky and Enteritidis serovars formed pellicle at liquid-air interface at 37°C. Three different colony morphotypes (saw; smooth and white, bdar; brown, dry and rough, rdar; red, dry and rough) were determined on Congo Red Agar among the isolates. High biofilm formation abilities of the tested Salmonella isolates can lead to widespread of virulence and resistance properties, especially to medically important antibiotics such as ciprofloxacin, via food chain. This situation constitutes an important concern for public health.Öğe DETERMINATION OF in vitro BIOFILM FORMATION ABILITIES OF FOOD BORNE Salmonella enterica ISOLATES(Trakya Univ Balkan Yerlesesi Enstituler Binasi, 2019) Aksoy, DenizSalmonellosis caused by non-typhodial Salmonella enterica serotypes is one of the most important food-borne diseases worldwide and biofilm structure formed by these pathogens provide a reservoir for food contamination and a source for infections. This study was performed in order to determine biofilm formation abilities of food borne Salmonella isolates on polystyrene and on air liquid interphase and their colony morphologies when grown on Congo Red Agar plates. 32 food-borne Salmonella strains isolated from retail chicken carcasses in Edirne province of Turkey and belonging to the Infantis, Enteritidis, Kentucky and Telaviv serotypes were used. The microtiter plate technique was used to determine biofilm formation abilities of the isolates on polystyrene surfaces by measuring the optical density (OD) values of the stained bacterial biofilms. The results showed that the strongest biofilm formation capacities of the isolates were observed at 22 degrees C for 3 days of incubation. Although all isolates formed pellicle on the liquid-air interface at 22 degrees C, only 13% of the isolates belonging to the Infantis, Kentucky and Enteritidis serovars formed pellicle at liquid-air interface at 37 degrees C. Three different colony morphotypes (saw; smooth and white, bdar; brown, dry and rough, rdar; red, dry and rough) were determined on Congo Red Agar among the isolates. High biofilm formation abilities of the tested Salmonella isolates can lead to widespread of virulence and resistance properties, especially to medically important antibiotics such as ciprofloxacin, via food chain. This situation constitutes an important concern for public health.Öğe Determination of the Role of Salmonella enterica Plasmids in Antibiotic Susceptibility and Caenorhabditis elegans Pathogenicity(Ankara Microbiology Soc, 2018) Aksoy, Deniz; Sen, EcePoultry animals and poultry associated products are important risk sources for Salmonellosis. S.Kentucky and S.Infantis are among the serovars frequently isolated from retail chickens and were reported to be isolated in Turkey. In this study, the role of plasmids carried by S.Kentucky and S.Infantis isolates in antibiotic resistance profiles of the isolates and their pathogenicity on Caenorhabditis elegans nematode model system were investigated. The isolates used, 1 of Kentucky and 2 of Infantis serotypes, were selected among food-borne Salmonella isolated from chicken carcass in Edirne. All three isolates were previously shown to contain plasmids carrying multidrug resistance and were known to be pathogenic on C.elegans nematode model system. S.Kentucky A10 isolate was resistant to ampicillin, nalidixic acid, tetracycline, ciprofloxacin, trimethoprim and sulphonamide and carried one plasmid with 31.6 kb size. S.Infantis A15 isolate was resistant to ampicillin, streptomycine, nalidixic acid, tetracycline, neomycin, sulphonamide and kanamycin and carried a plasmid with 19.9 kb size while the other S.Infantis isolate (A16) was resistant to streptomycin, nalidixic acid, tetracycline, trimethoprim, neomycin, sulphonamide and kanamycin and carried 3 plasmids with 42.4, 1.5 and 1.2 kb sizes. Plasmid curing experiments were performed to investigate the role of plasmids in antibiotic resistance and pathogenicity in C.elegans. Ethidium bromide (EtBr) dye was used as the plasmid curing agent. Plasmids were isolated from cultures grown in LB broth with different concentrations of EtBr (50, 75, 100, 125 pg/ml) according to the Kado-Liu method and the most effective EtBr concentration was determined as 125 pg/ml. C.elegans pathogenicity assays were carried out using plasmid cured isolates. The time 50% of the nematode die (TD50) values of the nematode groups fed with plasmid cured isolates were compared with previously obtained TD50 values of the nematode groups fed with wild type Salmonella isolates. Student's t-test (p<0.05) was used to showthe level of significance between TD50 values of the two groups. TD50 values of the positive control group fed with S.Typhimurium ATCC 14028 and the negative control group fed with Escherichia coli OP50 were found as 4.0 +/- 0.4 and 8.0 +/- 0.02 days, respectively. The differences between TD50 values of nematode groups fed with wild type and plasmid cured isolates were statistically significant both for S.Kentucky (A10) (4.9 +/- 0.04-6.2 +/- 0.1) and S.Infantis (A16) (4.4 +/- 0.01-6.2 +/- 0.2) (p< 0.05) strains, but no significant difference was observed for the groups fed with wild type and plasmid cured S.Infantis (A15) (5.7 +/- 0.39-5.8 +/- 0.16) strain. Broth microdilution method was used to determine whether there was any change in minimal inhibitory concentrations (MIC) of the antibiotics for which the isolates were resistant before plasmid elimination. No significant difference was found between the MIC values of the resistant antibiotics among Salmonella isolates carrying plasmids and with cured plasmid. This study is important since the first in vivo results about the role of Kentucky and Infantis serovar plasmids on C.elegans nematode model system were presented.Öğe Gıda Kökenli Salmonella enterica Suşlarının Patojenik Fenotiplerinin ve Virülans Determinantlarının Caenorhabditis elegans Hayvan Modelinde İncelenmesi(2015) Aksoy, Deniz; Şen, EceTifo dışı Salmonella enterica serotipleri ile kontamine gıdaların tüketiminin neden olduğu salmonellozlar, gıda kökenli hastalıkların başında gelmekte ve bu durum mikrobiyal gıda güvenliğini, önemli bir halk sağlığı sorunu haline getirmektedir. Bu çalışma, Edirne ilindeki gıda kaynaklı Salmonella enterica suşlarının antibiyotik dirençlerinin, serotiplerinin, plazmid profi llerinin ve Caenorhabditis elegans hayvan modeli sisteminde patojenite potansiyellerinin belirlenmesi amacıyla gerçekleştirilmiştir. Çalışmada, tavuk karkaslarından izole edilmiş ve 26'sı (%81.25) Infantis, dördü (%12.5) Enteritidis, birer tanesi de (%3.1) Telaviv ve Kentucky serotiplerine dahil olan 32 Salmonella izolatı kullanılmıştır. İzolatların antibiyotik direnç profi lleri, disk difüzyon ve buyyon mikrodilüsyon yöntemleriyle belirlenmiştir. İzolatların patojenite potansiyellerinin belirlenmesi amacıyla yeni bir C.elegans nematod model sistemi kullanılmıştır. Antibiyotik direnç profi llerine göre 32 suştan birinin (%3) gentamisine, ikisinin (%6.2) siprofl oksasine, üçünün (%9.4) ampisiline, 18'inin (%56.3) kanamisine, 19'unun (%60.8) neomisine, 25'inin (%78.1) tetrasikline, 25'inin (%78.1) trimetoprime, 26'sının (%81.2) nalidiksik aside, 27'sinin (%84.4) streptomisine ve tümünün (%100) sülfonamid bileşiklerine dirençli olduğu belirlenmiştir. Çalışılan 32 suşun hepsi kloramfenikol ve ampisillin/sulbaktama karşı duyarlı bulunmuştur. Streptomisin, nalidiksik asit, tetrasiklin, trimetoprim, sülfonilamid bileşikleri, kanamisin ve neomisine karşı yüksek düzeyde direnç belirlenmiştir. Plazmid analizi sonuçlarına göre altı izolatın (%18.75), büyüklükleri 1.2 ile 42.4 kb arasında değişen 1-3 adet plazmid taşıdığı belirlenmiştir. C.elegans nematod modeli sisteminde, her bir deney grubu için nematodların %50'sinin ölmesi için geçen gün (TD50) hesaplanmıştır. TD50 değerleri, pozitif kontrol olarak kullanılan S.Typhimurium ATCC 14028 ile beslenen nematod grubu için 4.2 ± 0.5 gün, negatif kontrol olarak kullanılan E.coli OP50 ile beslenen nematod grubu için ise 8.0 ± 0.02 gün olarak hesaplanmıştır. TD50 değeri, Salmonella izolatları ile beslenen nematod grupları için ise 3.4 - 7.3 gün arasında bulunmuştur. Pozitif kontrol grubundan elde edilen TD50 değeri ile deney gruplarından elde edilen TD50 değerleri arasındaki farklılığın istatistiksel olarak anlamlı olup olmadığı Student's t-testi ile analiz edilmiştir. Infantis serotipinden altı ve Enteritidis serotipinden dört adet olmak üzere toplam 10 izolatın (%31.25) C.elegans için patojen olmadığı; Infantis, Kentucky ve Telaviv serotiplerine ait izolatları içeren diğer 22 izolatın (%67.75) ise patojen olduğu belirlenmiştir (p< 0.05). Patojen izolatlardan 20'sinde (%90.9) çoklu ilaç direnci saptanmış ve üçünün, büyüklükleri 1.2 ile 42.4 kb arasında değişen 1-3 adet plazmid taşıdığı tespit edilmiştir. Elde edilen sonuçlar, antibiyotik direnci taşıyan Salmonella enterica suşlarının yaygınlığını vurgulamış ve yeni deney hayvanı enfeksiyon modelleri sayesinde çevresel ve gıda kökenli suşların patojenik potansiyellerinin incelenmesini hedefl eyen çalışmalara pratik bir yaklaşım sağlamıştır. Bu çalışmada, C.elegans, yurdumuzda ilk kez gıda kökenli Salmonella serotiplerinin patojenite profi llerinin saptanmasında kullanılmıştırÖğe Investigation of Pathogenic Phenotypes and Virulence Determinants of Food-Borne Salmonella enterica Strains in Caenorhabditis elegans Animal Model(Ankara Microbiology Soc, 2015) Aksoy, Deniz; Sen, EceSalmonellosis, caused by non-typhoidal Salmonella enterica serovars with the consumption of contaminated food, is one of the leading food-borne disease that makes microbial food safety an important public health issue. This study was performed in order to determine the antibiotic resistance, serotyping, plasmid profiles and pathogenicity potentials of food-borne Salmonella isolates in Caenorhabditis elegans animal model system in Edirne province, located at Thrace region of Turkey. In this study, 32 Salmonella isolates, of which 26 belonged to Infantis, four to Enteritidis, one to Telaviv and one to Kentucky serovars, isolated from chicken carcasses were used. Antibiotic resistance profiles were determined by disc diffusion and broth microdilution methods. A new C.elegans nematode animal model system was used to determine the pathogenicity potential of the isolates. The antibiotic resistance profiles revealed that one (3.1%) isolate was resistant to gentamicin, two (6.2%) to ciprofloxacin, three (9.4%) to ampicillin, 18 (56.3%) to kanamycin, 19 (60.8%) to neomycin, 25 (78.1%) to tetracycline, 25 (78.1%) to trimethoprim, 26 (81.25%) to nalidixic acid, 27 (84.4%) to streptomycin and 32 (100%) to sulfonamide. All of the 32 strains were susceptible to chloramphenicol and ampicillin/sulbactam. High levels of resistance to streptomycin, nalidixic acid, tetracycline, trimethoprim, sulfonamide, kanamycin and neomycin was determined. According to the plasmid analysis, six isolates (18.75%) harboured 1-3 plasmids with sizes between 1.2 and 42.4 kb. In C.elegans nematode animal model system, the time (in days) required to kill 50% (TD50) of nematodes was calculated for each experimental group. TD50 values of the nematode group fed with S.Typhimurium ATCC 14028 that was used as the positive control and another group fed with E.coli OP50 as the negative control were 4.2 +/- 0.5 days and 8.0 +/- 0.02 days, respectively. TD50 of the groups fed with Salmonella isolates ranged between 3.4 and 7.3 days. The significance of the differences between TD50 values of the positive control and experimental groups was analysed by using Student's t test. Ten of the isolates (31.25%), of which six belonged to Infantis and four to the Enteritidis serotypes were non-pathogenic, and the rest 22 isolates including Infant's, Kentucky and Telaviv serovars (67.75%) were found to be pathogenic for the C.elegans animal system (p<0.05). Twenty of the isolates (90.9%) which were determined as pathogens showed multiple drug resistance and three of them possessed 1-3 plasmids, sizes between 1.2 - 42.4 kb. The overall results underlined wide distribution of antibiotic-resistant Salmonella enterica strains and provided a practical alternative for studies aiming determination of pathogenic potential of environmental and food-borne strains through new experimental animal infection model. In this study, C.elegans was utilized for the first time to determine the profiles of pathogenicity of food-borne Salmonella serotypes in Turkey.Öğe Salmonella spp. kökenlerinin patogenez determinantları ve salmonellozların moleküler tanısında kullanımları(Trakya Üniversitesi Fen Bilimleri Enstitüsü, 2015) Aksoy, Deniz; Şen, EceBu çalışma, Edirne ilindeki tavuk karkaslarından izole edilmiş olan Salmonella izolatlarının patojenite fenotiplerinin saptanması ve patogenez determinantlarının belirlenmesi amacı ile yapılmıştır. Toplam 32 izolat bu çalışmaya dahil edilmiştir ve izolatlardan 26’sı Infantis, 4’ü Enteritidis, 1’er tanesi de Telaviv ve Kentucky serotiplerine dahildir. İzolatların tamamında kullandığımız antibiyotiklerden en az bir tanesine karşı direnç ve 26 tanesinde çoklu ilaç dirençliliği gözlemlenmiştir. Plazmid analizi sonuçlarına göre 6 izolatın, büyüklükleri 1.2-42.4 kb arasında değişen 1-3 plazmid taşıdığı saptanmıştır. Infantis serotipinden 6 ve Enteritidis serotipinden 4 tane olmak üzere toplam 10 izolatın Caenorhabditis elegans için patojen olmadığı, Infantis,Kentucky ve Telaviv serotiplerine ait izolatları içeren diğer 22 izolatın ise patojen olduğu belirlenmiştir (p<0.05). BALB/c fare model sisteminde ise, Infantis serotipinden 3 ve Enteritidis serotipinden 1 izolatın patojen olmadığı ve Kentucky, Telaviv ve Enteritidis serotipinden 1'er izolatın patojen olduğu tanımlanmıştır (p<0.05). Plazmid giderme çalışmalarında antibiyotik direnci ile plazmidler arasında bir ilişkiye rastlanamazken plazmid kaybının C. elegans'ta patojenitenin azalmasına yol açtığı bulunmuştur. mRNA analizi çalışmalarında fim ve stn geninin fare ve nematod modelinin her ikisinde de eksprese edilmemiş, patojenite ile ilişkili olduğu bilinen invA geni ise sadece nematod modelinde eksprese edilmiştir. İki hayvan modeli sisteminde Salmonella izolatlarının patojenite fenotipleri ve virülans gen anlatımlarının farklı olduğu tespit edilmiştir. Tıbbi DNA tabanlı tanı kiti geliştirmek için hasta izolatlarının kullanılması gerekmektedir.
