[Determination of the role of Salmonella enterica plasmids in antibiotic susceptibility and Caenorhabditis elegans pathogenicity]. / Salmonella enterica plazmitlerinin antibiyotik duyarliligi ve Caenorhabditis elegans patojenitesindeki rollerinin belirlenmesi.

Poultry 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 µg/ml) according to the Kado-Liu method and the most effective EtBr concentration was determined as 125 µg/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. Studentðs 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.

Detection and Characterization of the Emerging Relapsing Fever Pathogen, Borrelia miyamotoi, from the Ixodes ricinus Tick in the Rural Trakya (Thrace) Region of Northwestern Turkey.

The hard tick-borne relapsing fever agent, Borrelia miyamotoi infection in Ixodes ricinus ticks sampled from Istanbul and the countryside of Kirklareli in northwestern Turkey, was examined by TaqMan-PCR targeting 16S rDNA, nested PCR targeting 16S rDNA, the flagellin gene (flaB), and the 16S and 23S rDNA intergenic spacer (IGS), and sequencing analyses of these amplicons. B. miyamotoi was detected in 1 out of 248 I. ricinus ticks (infection rate 0.4%). The tick infected with B. miyamotoi was collected in Longos, Kirklareli province on the European side of Turkey near the Bulgarian border. The 16S rDNA, flaB, and IGS sequences from the infected tick showed high similarities to those of B. miyamotoi detected in I. ricinus in Europe.

[Investigation of antitumorigenic effects of food-borne non-pathogenic and pathogenic Salmonella enterica strains on MEF, DU145 and HeLa cell lines]. / Gida kökenli patojen olmayan ve patojenik Salmonella enterica suslarinin antitümörijenik etkilerinin MEF, DU145 ve HeLa hücre kültürlerinde incelenmesi.

Basic applications in cancer therapy may fail to eradicate cancer cells completely, they can show toxic affects to healthy cells and development of resistance to antitumor agents may increase tendency to metastasis. Bacterial therapies have the advantage of specific targetting of tumors by selective toxicity, responsiveness to external signals, self-propelling capacity, and the sense of microenvironment. The most interest on the bacterial cancer therapy is about Salmonella spp. with a special emphasis of S.Typhimurium. The aim of this study was to investigate the antitumorigenic effects of food-borne non-pathogenic and pathogenic Salmonella enterica strains on different cell cultures. Non-pathogenic Salmonella Enteriditis (A17) and pathogenic Salmonella Telaviv (A22) strains isolated from chicken carcasses which were put on the market in Edirne province (located at Thrace region of Turkey), and Salmonella Typhimurium ATCC 14028 strain were used in the study. ATCC-derived MEF (mouse embryonic fibroblasts), DU145 (human prostate cancer cells), and HeLa (human cervical cancer cells) cell lines were cocultivated with Salmonella strains of MOI (Multiplicity of infection; number of bacteria:number of cell) of 1000:1, 100:1, 10:1, 1:1, 0.1:1. The cell viability was measured by colorimetric MTT cytotoxicity assay, the percentage of apoptosis was assessed by Tali® Apoptosis Assay-Annexin V Alexa Fluor® 488 kit (Invitrogen, Molecular Probes, Life Technologies, USA), and the caspase-3 activity was determined by colorimetric protease ApoTarget™ kit (Invitrogen, BioSource International, USA). It was shown that non-pathogenic S.Enteriditis (A17) decreased cell viability approximately to 70%, wheras patogenic S.Telaviv (A22) and standart S.Typhimurium ATCC 14028 strains reduced cell viability approximately to 80%. Adversely, it was also observed that pathogenic S.Telaviv (A22) strain induces apoptosis more effectively than non-pathogenic S.Enteriditis (A17) and S.Typhimurium ATCC 14028 strains. Apoptosis percentage induced by pathogenic S.Telaviv (A22) strain was approximately 15% while 5% for both non-pathogenic S.Enteriditis (A17) and S.Typhimurium ATCC 14028 strains. Similarly, average OD405 values of caspase-3 activity was shown as 0.01 for both non-pathogenic S.Enteriditis (A17) and S.Typhimurium ATCC 14028 strains whereas average OD405 value of caspase-3 activity for pathogenic S.Telaviv (A22) strain was very close to 0.02 and it doubled the value for negative control. Our data are important in terms of the indication of food-borne pathogenic S.Telaviv (A22) strain that enhanced caspase-3 activity and induced apoptosis, and S.Enteriditis (A17) strain that showed selective cytotoxicity on DU145 (human prostate cancer cells).

[Investigation of pathogenic phenotypes and virulence determinants of food-borne Salmonella enterica strains in Caenorhabditis elegans animal model]. / Gida kökenli Salmonella enterica suslarinin patojenik fenotiplerinin ve virülans determinantlarinin Caenorhabditis elegans hayvan modelinde incelenmesi.

Salmonellosis, 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 Infantis, 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.

Enhanced Adhesion and OspC Protein Synthesis of the Lyme Disease Spirochete Borrelia Burgdorferi Cultivated in a Host-Derived Tissue Co-Culture System.

BACKGROUND: The adhesion process of Borrelia burgdorferi to susceptible host cell has not yet been completely understood regarding the function of OspA, OspB and OspC proteins and a conflict exists in the infection process. AIMS: The adhesion rates of pathogenic (low BSK medium passaged or susceptible rat joint tissue co-cultivated) or non-pathogenic Borrelia burgdorferi (high BSK medium passaged) isolate (FNJ) to human umbilical vein endothelial cells (HUVEC) cultured on coverslips and the synthesis of OspA and OspC proteins were investigated to analyze the infection process of this bacterium. STUDY DESIGN: In-vitro study. METHODS: Spirochetes were cultured in BSK medium or in a LEW/N rat tibiotarsal joint tissue feeder layer supported co-culture system using ESG co-culture medium and labelled with 3H-adenine for 48 hours. SDS-PAGE, Western Blotting, Immunogold A labeling as well as radiolabeling experiments were used to compare pathogenic or non pathogenic spirochetes during the adhesion process. RESULTS: Tissue co-cultured B. burgdorferi adhered about ten times faster than BSK-grown spirochetes. Trypsin inhibited attachment to HUVEC and co-culture of trypsinized spirochetes with tissues reversed the inhibition. Also, the synthesis of OspC protein by spirochetes was increased in abundance after tissue co-cultures, as determined by SDS-PAGE and by electron microscopy analysis of protein A-immunogold staining by anti-OspC antibodies. OspA protein was synthesized in similar quantities in all Borrelia cultures analyzed by the same techniques. CONCLUSION: Low BSK passaged or tissue co-cultured pathogenic Lyme disease spirochetes adhere to HUVEC faster than non-pathogenic high BSK passaged forms of this bacterium. Spirochetes synthesized OspC protein during host tissue-associated growth. However, we did not observe a reduction of OspA synthesis during host tissue co-cultivation in vitro.

Molecular detection of Anaplasma phagocytophilum and Borrelia burgdorferi in Ixodes ricinus ticks from Istanbul metropolitan area and rural Trakya (Thrace) region of north-western Turkey.

We demonstrated the presence of the agent of human granulocytic anaplasmosis (HGA), Anaplasma phagocytophilum, and the agent of Lyme borreliosis, Borrelia burgdorferi sensu lato, in north-western Turkey. A total of 241 questing Ixodes ricinus adult ticks were sampled by flagging from recreational parks of the Asiatic and European sides of the heavily populated Istanbul metropolitan area and rural forests of Kirklareli located in the Thrace region in 2008. Both tick-borne pathogens were detected and identified by PCR and DNA sequencing analysis. A. phagocytophilum infection rates were 2.7% in Istanbul and 17.5% in the Kirklareli area. B. burgdorferi sensu lato infection rates were 38.7% in Istanbul and 11.4% in the Kirklareli area. Only 3 of 241 ticks were coinfected with A. phagocytophilum and B. burgdorferi sensu lato.

Isolation of moderately infectious Borrelia burgdorferi sensu stricto from attenuated cultures by using complement-mediated, antibody-dependent lysis selection technique in a mammalian tissue co-culture system.

We investigated the association between complement resistance and phenotypes of pathogenicity of Borrelia burgdorferi sensu lato isolates cultivated in a LEW/N rat tibiotarsal joint-derived tissue feeder layer-supported co-culture system. Guinea pig complement and immune serum raised in LHS/Ss hamsters caused complete lysis of B. burgdorferi sensu stricto isolate 297, B. afzelii and B. garinii in Barbour-Stoenner-Kelly's medium; however, tissue co-cultured B. burgdorferi sensu stricto contained complement escape variants. The arthritogenicity and infectivity of these variants were tested in 3-week-old Syrian hamsters and in a vaccinated hamster model in which formalin-killed B. burgdorferi sensu stricto C-1-11 vaccinated animals develop severe arthritis after challenge with live, pathogenic, low-passage 297 isolate. Non-animal-passaged complement escape variants were infectious in both animal models as demonstrated by re-isolation from the infected animals and competitive PCR. IP injection of animal-passaged complement escape variants caused development of severe arthritis in vaccinated animals 5 weeks post-injection; animal passage of complement escape variants was necessary for isolation of arthritogenic spirochetes from high-passaged, non-arthritogenic, attenuated borrelia cultures. Complement escape variants synthesized outer surface protein E as demonstrated by SDS-PAGE and western blotting analyses. The complement-mediated selection technique in tissue co-culture provides a novel approach to the studies of Lyme disease, enables us to isolate pathogenically distinct borrelia populations from attenuated cultures and prepare a moderately infectious, non-pathogenic live vaccine against this illness.