[Galleria mellonella larva model in evaluating the effects of biofilm in Candida albicans]. / Candida albicans biyofilm etkilerinin degerlendirilmesinde Galleria mellonella larvasi modeli.

Biofilm-related infections are chronic infections that cause serious increase in morbidity and mortality as well as significant economic loss. Galleria mellonella larva is shown as a reliable animal model for in vivo toxicology and pathogenicity tests due to its large size, ease of practice, ability to survive at 15-37°C and its similarity to mammals' natural immune system. The aim of this study was to evaluate the effects biofilm activity of Candida albicans in a G.mellonella larva model. Two C.albicans strains isolated as a disease agent were used for the model, where one was positive (BP), and the other one was negative (BN) for biofilm production. Eighty healthy G.mellonella larvae, all in the last larval stage and 2-2.5 cm long, were divided into 4 groups of equal size. Group 1 was set as the control group. Group 2 was injected with sterile phosphate buffer (PBS) group. Group 3 was injected with BP C.albicans strain and group 4 with BN C.albicans strain. A 5 µL volume of C.albicans prepared at 5 × 105 cfu/ml concentration with PBS was injected into the last left rear-legs of the larvae. The larvae were kept in sterile petri dishes at 37°C. They were observed for a total of 96 hours, for 4 hours in the first 24 hours, then in 12 hours intervals. Melanization, survival, total hemocyte count and fungal burden were evaluated as infection indicators. Melanization and death were not observed throughout the study period in group 1. One larva died in group 2. Small melanization spots (dark spots) and subsequent progressive melanization were observed from 3rd hour in the larvae infected with C.albicans. When compared with the BN C.albicans infected group, survival rate was 20% for BP C.albicans infected larvae at the end of 24 hours. Total hemocyte count was very low in the infected groups compared to groups 1 and 2, also significantly lower in group 3 than in group 4. In quantitative cultures, growth of C.albicans was detected in groups 3 and 4 while not in groups 1 and 2. Fungal load was significantly higher in BP C.albicans infected group than BN C.albicans infected group. In this study, G.mellonella larvae were used as live hosts to demonstrate the effects of biofilm activity of C.albicans. Our results suggest that larval models can be used to investigate the effects of fungal infections and biofilm like virulence factors on host cells, and invertebrate animal models can be widely used and can bridge between in vitro studies and mammalian models.

[Evaluation of the Relationship Between Microenvironment and Biofilm and Coagulase Responses of Staphylococcus aureus Strains Under the Effect of Gentamicin]. / Gentamisin Etkisi Altindaki Staphylococcus aureus Suslarinin Biyofilm ve Koagülaz Yanitlari ile Mikroçevre Iliskisinin Degerlendirilmesi.

The frequency of infections caused by Staphylococcus aureus strains and the rate of antibiotic resistance among these strains are gradually increasing. Accordingly, serious problems emerge in the treatment of community or hospital acquired S.aureus infections. This study was aimed to determine the role of MIC and sub-MIC concentrations of gentamicin on biofilm and coagulase forming effects of S.aureus in in vitro test systems and cell cultures. A standard S.aureus ATCC 25923 strain and two clinical S.aureus strains isolated from blood cultures (C1 and C2) were included in the study. Gentamicin MIC values of the strains were determined with microdilution method at the cation-adjusted Mueller Hinton broth according to CLSI standards. For each strain, MIC, 50% MIC and 25% MIC values of gentamicin were determined separately. At the determined MIC values, biofilm formations of strains were determined with crystal violet method spectrophotometrically. Also, coagulase activities of the strains were evaluated in glass tubes. Human origin epithelial cell cultures namely HEp-2 cell lines, were infected with the standard and clinical S.aureus strains (Multiplicity of infection: 50/1) and left for incubation for two hours. After all, MIC, 50% MIC and 25% MIC values of gentamicin, were added to infected cell lines and incubated for 18 hours. Cells were blown up with distilled water and then bacteria were collected. Biofilm formation and coagulase production of these bacteria were evaluated. When S.aureus ATCC 25923 strain and C1 strains' biofilm formation was evaluated before (in vitro) and after incubation in cell culture, no difference was observed. However in C2 strain, under the effect of MIC level gentamicin, biofilm formation was occurred after interaction with the cell. In the same way, when coagulase responses were evaluated, after interaction with the cell, coagulase production of C2 strain was inhibited. These results indicated that, phenotypic characteristics such as biofilm formation and coagulase production might change during the process of bacterial adaptation to microenvironment. Further advanced experimental modelling designed with different combinations of antibiotics and different cell lines may provide data about the causes and timing of these phenotypic changes and shed light on the development of new treatment policies.

[Experimental oral candidiasis in healthy and immunocompromised BALB/c mice]. / Saglikli ve Immün Sistemi Baskilanmis BALB/c Farelerde Deneysel Oral Kandidiyaz.

Oral candidiasis which is the most common type of Candida infections affecting humans, is most frequently caused by C.albicans. Immune response of the host, as well as a variety of virulence factors of the causative agent, play important roles in the development of Candida infections. The colonization rate of Candida in the oral cavity of healthy individuals, is between 25-30%, however, this rate is reported to be increased in immunosuppressive subjects. In our study, we established an oral candidiasis model with C.albicans in healthy and experimentally immunocompromised mice and aimed to compare Candida colonization rates and histopathological changes occurred in the tongue and esophagus tissues of the animal groups. A total of 21 BALB/c mice were grouped as control (Group 1; n= 7), healthy (Group 2; n= 7) and immunocompromised (Group 3; n= 7) groups. Immunosuppression in mice was performed by subcutaneous injection of prednisolone. For experimental oral candidiasis, cotton swab impregnated with C.albicans strains which did not have acid proteinase and phospholipase enzyme activity, no biofilm production, and sensitive to fluconazole and amphotericin B, were used. In the control group, physiological saline solution was used instead of C.albicans strain. In the forth day of experimental oral candidiasis model swab samples taken from the dorsal tongue surface of mice were evaluated by quantitative cultivation method. No yeast colonies were detected in Group 1 while more significant number of yeast colonies were observed in Group 3 compared to Group 2 (p= 0.002). Tongue and esophagus tissues of mice were stained with hematoxylin-eosin and periodic acid schiff staining and evaluated in terms of inflammatory response, abscess formation, vascular congestion, vasodilation and for the presence of yeast and hyphae. When the inflammation in esophagus was considered, statistically significant difference was determined between group 1 and group 3 (p= 0.023), however, no difference was detected between group 2 and 3 (p= 0.107). The level of inflammation in tongue tissue exhibited no difference between groups 2 and 3 (p= 0.317) while the difference was significant when these groups were compared to the control group (p= 0.00, p= 0.002, respectively). Similarly, the level of congestion in tongue tissue exhibited no difference between groups 2 and 3, however, the difference was significant when compared to the control group. To enlighten the relation between host immune status and oral candidiasis caused by C. albicans, further larger-scale studies also concerning the various virulence factors of the infectious agent, should be conducted by the use of experimental animal models which may successfully guide us in this regard.

[Comparison of different antifungal treatment regimens for experimental Candida endophthalmitis in rabbit models]. / Tavsanlarda olusturulan deneysel Candida endoftalmisinde farkli tedavi seçeneklerinin karsilastirilmasi.

The aim of this study was to evaluate the treatment options of experimental in-vivo Candida endophthalmitis. For inoculation, a 0.1 ml of suspension of Candida albicans was injected into the vitreous of the right eye of each New Zealand rabbit. On the 15th day, the clinical evaluation for the resultant endophthalmitis was noted, and vitreous samples were obtained. On the 21st day, culture positive eyes were divided into four groups in terms of treatment modalities. Group 1 (n = 7) received intravitreal amphotericin B injection, group 2 (n = 8) received both intravitreal dexamethasone and amphotericin B injections, group 3 (n = 8) underwent pars plana vitrectomy (PPV) and amphotericin B injection, and group 4 (n = 8) underwent PPV and both amphotericin B and silicone oil injections. The vitreous samples obtained from right eyes of the rabbits on the 15th day, were all culture positive for Candida albicans. On the 35th day, the least colony counts (colony forming unit) were present in eyes that received only intravitreal amphotericin B injection in group 1, followed by group 4 that underwent PPV and both amphotericin B and silicone oil injections. In Candida endophthalmitis, intravitreal injection of amphotericin B without steroid appears to be the primary choice of therapy. In cases who fail to respond to this regimen alone, PPV in combination with silicone oil injection may be considered. Benefit-risk ratio should be cautiously interpreted for application of intravitreal steroid injection.

Effects of non toxic doses of acyclovir on nitric oxide and cellular death responses in herpesvirus types 1 and 2 infected hep-2 cells.

Herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) are among the most "successful" pathogens and code for a variety of proteins to direct the apoptosis/necrosis responses of the cells they infect. Nitric oxide (NO) is an important intracellular signaling molecule in pathological processes. Acyclovir (ACV) is a chain terminator that targets the viral DNA polymerase as an antiviral agent. In this study, NO signals, and apoptosis/necrosis responses of HEp-2 cells were compared when infected by HSV-1 and -2 for 24 hours against non toxic doses (starting from 48.8, 24.4, 12.2, 6.1, 3 to 1.5 microg/mL) of ACV. In 48.8, 24.4 and 12.2 microg/mL of ACV, HSV-1 had an "upregulating effect" whereas HSV-2 had a "downregulating effect" on NO production, and in 6.1, 3 and 1.5 microg/mL of ACV HSV-1 had a "down-regulating effect" whereas HSV-2 had an "upregulating effect" on NO responses (HSV-1 had a "downregulating effect" on NO production whereas HSV-2 had an "upregulating effect" on NO production without any ACV). In 48.8, 24.4 and 12.2 microg/mL of ACV, HSV-1 had an "anti-apoptotic effect" whereas HSV-2 had a stimulation on "apoptotic effect", and in 6.1, 3 and 1.5 microg/mL of ACV HSV-1 had an "apoptotic effect" and HSV-2 turned to "its natural viral apoptotic effect level" (HSV-1 had an "natural viral apoptotic effect" whereas HSV-2 had a "natural viral apoptotic effect" on apoptosis response without any ACV). In 48.8, and 24.4 microg/mL of ACV, HSV-1 had significant "necrotic effect" on necrotic cellular death, "necrosis" increased in 12.2, 6.1, 3 and 1.5 microg/mL of ACV (HSV-1 had a negligible "necrotic effect" on HEp-2 cells alone), and HSV-2 had a "natural viral necrotic effect" alone; and also in all non toxic ACV concentrations. These results showed that HSV-1 and -2 had different "strategies" on apoptosis/necrosis and NO with and without non toxic ACV. These differences deserve further studies in order to explain the interactions between apoptotic/anti apoptotic, necrotic genes and NO, and ACV in HSV-1 and HSV-2 infections respectively.

Molecular characterization of TEM- and SHV-derived extended-spectrum beta-lactamases in hospital-based Enterobacteriaceae in Turkey.

TEM- and SHV-derived extended-spectrum beta-lactamases (ESBLs) producing Enterobacteriaceae have been reported from throughout the world, but there has been limited data for the molecular characterization of these enzymes in Turkey. The aim of this study was to investigate and to type the TEM- and SHV-derived ESBLs in 63 ESBL-producing clinical isolates of Enterobacteriaceae, and it included further analysis; transfer experiments, isoelectric focusing, PCR, PCR-restriction fragment length polymorphism, and DNA sequencing. According to PCR results the transconjugant strains included 52.7% TEM, 74.3% SHV, and 32.4% of both the TEM and SHV genes. Using PCR/NheI restriction analysis, 45 of the 46 ESBL detected in transconjugants were determined to be SHV-derived. DNA sequencing was performed for the identification of TEM- and SHV-derived ESBLs for 18 selected transconjugants. SHV-2, SHV-5, and SHV-12 were detected in five, seven, and five samples, respectively. This is the first description of SHV-12 in Turkey.

[The importance of antinuclear (ABA) and anti-double stranded dna (anti-dsDNA) antibodies in the diagnosis of connective tissue diseases.]. / Konnektif doku hastaliklarinin tanisinda antinükleer (ANA) ve anti-double stranded dna (anti-dsDNA) antikorlarinin önemi.

Connective tissue diseases (CTD) are characterized by the presence of autoantibodies against several tissues. These autoantibodies occur against cell membrane, cell receptors, plasma proteins, and cytoplasmic and nuclear components. In laboratories, anti-nuclear antibodies (ANA) and anti-double stranded DNA (anti-dsDNA) antibodies are widely used in diagnosis of CTD. The aim of this study was to investigate the presence and accompaniment of ANA and anti-dsDNA antibodies in diagnosis of several CTD and also to study the prevalence of ANA and anti-dsDNA in a group of 88 patients with various types of CTD. ANA were detected by immunofluorescence (IFA) using HEp-2 cells (Zeus Scientific, Inc. USA) and anti-dsDNA antibodies using Crithidia luciliae (BioSystems, Spain) as substrates in immunofluorescence. ANA Western Blot (WB) Immunoassay (ImmuBlot, International Immuno-Diagnostics, USA) was also used along with the tests referred to previously. ANA was found in the sera of 84 (96.5%) patients while anti-dsDNA was detected in 7 (7.95%). Moreover different fluorescence patterns were also evaluated with ANA IFA in accordance with anti-dsDNA results. Mixed patterns in three and a homogeneous pattern in four anti-dsDNA positive patients' sera were determined on HEp-2 cell line by IFA. Seven sera which were ANA and anti-dsDNA positive with IFA were also found to be positive with WB and their ANA patterns with the specific ANA WB bands were also evaluated. It was observed that IFA results were in concordance with WB results. Our data indicated that the above findings should be controlled and evaluated with a more advanced method such as western blotting technique in order to confirm the presence of specific antibodies along with clinical outcome of the patients. As a result we think that ANA WB method is an appropriate technique in diagnosis of CTD as anti-dsDNA and ANA bands can be evaluated together with this method.

[The importance and validity of anti-Toxoplasma gondii IgG, IgM, IgA antibodies and IgG avidity tests in the diagnosis of Toxoplasmosis infection during pregnancy.]. / Gebelikte Toxoplasmosis tanisinda anti-Toxoplasma gondii IgG, IgM, IgA antikor ve IgG avidite testlerinin birlikteligi ve önemi.

Detection of specific IgG, IgM and IgA antibodies by enzyme immunoassay (EIA) tests are not always sufficient in the diagnosis of early and late Toxoplasma gondii infection during pregnancy. For this reason, the specific IgG avidity test should be used to detect primary toxoplasmosis infection. In this study, an investigation was made of the serological status of pregnant women who were suspected of having primary or late toxoplasmosis as well as the importance and relationship of specific anti-Toxoplasma gondii IgG, IgM and IgA antibodies and specific IgG avidity in their sera. TORCH panels were also used for these patients. A total of 52 pregnant women who were admitted in the Dokuz Eylül University Gynecology and Obstetrics Clinic were included in this study. The sera were sent to the serology and immunology laboratory for investigation of the anti-Toxoplasma gondii IgG, IgM and IgA antibodies by the EIA (Cobas Core, Roche, Germany and ETI-TOXOK-A, DiaSorin, Germany) technique. The anti- Toxoplasma gondii IgG avidity test was performed on IgG as well as IgM and/or IgA in positive sera with the EIA (Toxoplasma IgG Avidity EIA Well- RADIM, Italia) technique. The anti-Toxoplasma gondii IgG avidity test was not performed on sera from 21 pregnant women negative for anti-Toxoplasma gondii IgG, IgM and IgA antibodies. Borderline and high IgM levels showed a good correlation with the IgG avidity results. The ratio for the IgM and IgA positivity was 32.3% and both the IgM positivity and IgA negativity were 29%. In conclusion, anti- Toxoplasma gondii IgG avidity and anti-Toxoplasma gondii IgM antibody tests should be used together. However, positivity or high avidity of IgA was limited in the diagnosis of active toxoplasmosis infection of pregnant women.

[Comparison of indirect immunofluorescence and enzyme immunoassay methods for the determination of antinuclear antibodies]. / Antinükleer antikorlarin saptanmasinda indirek immünofloresan ve enzim immün yöntemlerinin karsilastirilmasi.

Antinuclear antibodies (ANA) are widely used for screening and monitoring of connective tissue diseases (CTD). Indirect immunofluorescence assay (IFA) is the standard method which is more often preferred for detecting these antibodies. Another method is enzyme immunoassay (ELISA) which includes extractable nuclear antigens (ENA). The aim of this study was to compare two different methods in view of their performances in the detection of ANA. A total of 27 sera from patients prediagnosed as different types of CTD, were screened by ANA-IFA (Zeus Scientific Inc, USA) and ELISA (Zeus Scientific Inc, ENA Profile-6, USA) methods. In addition, specific staining patterns of ANA on HEp-2 cells as a substrate, were enrolled with IFA. As a result, ANA positivity was detected in all of the 27 samples (100%) by IFA, and only in 7 (25.9%) by ELISA. The concordance rate between two different assays was estimated as 38.6%, and a statistically significant difference was found between the methods in the detection of ANA (x2=20, p<0.001). In conclusion, ANA-IFA method is still a reliable routine screening method for the laboratory diagnosis of CTD, while ENA Profile-6 ELISA may give false negative results, because of its limited antigen content, and should be supported with additional antigens.

Effect of subminimal inhibitory concentrations of three fluoroquinolones on adherence of uropathogenic strains of Escherichia coli.

The effect of subinhibitory concentrations (1/2-1/32 x MIC) of ciprofloxacin, ofloxacin and levofloxacin on the adherence of three strains of Escherichia coli (a mannose-resistant haemagglutinating clinical isolate, a non-haemagglutinating clinical isolate and the mannose-resistant haemagglutinating ATCC 25922 strain) were studied. Ciprofloxacin had the lowest MIC values but only the 1/2 MIC concentration inhibited adherence of mannose-resistant haemagglutinating strains after exposure to subMIC values. Significant inhibition of adherence was observed with 1/4 x MIC ofloxacin for both haemagglutinating isolate (27096) and the ATCC strain. Levofloxacin might be more effective and safer than ciprofloxacin and ofloxacin as a long acting fluoroquinolone at subMIC values in patients with UTI.