[Existence of Biofilm and Biofilm-Associated Virulence Genes in Multi-Drug Resistant Invasive Acinetobacter baumannii Isolates]. / Çok Ilaca Dirençli Invaziv Acinetobacter baumannii Izolatlarinda Biyofilm ve Biyofilm Iliskili Virülans Genlerinin Varligi.

Acinetobacter baumannii is a multi-drug resistant (MDR) gram-negative pathogen leading to nosocomial infections. Hospital-acquired infections due to A.baumannii occur especially in patients hospitalized in intensive care units. Important infections related to this bacterium are pneumonia, bacteremia, endocarditis, skin and soft tissue, urinary tract infections and meningitis. Human transmission is usually through the hospital environment or through medical personnel. A.baumannii isolates increases their virulence not only being multiple resistance to antibiotics but as well as the ability to form biofilm. The biofilm formation of A.baumannii isolates were mostly related with genes encoding curli fiber (csgA), the chaperone-usher fimbria (csuE) and the outer membrane (ompA). The aim of this study was to demonstrate biofilm production and virulence genes in MDR invasive A.baumannii isolates. MDR and similarity status previously known invasive A.baumannii (n= 156) isolates were included in the study. Biofilm production was determined by quantitative microplate biofilm method. Virulence genes csgA, csuE, fimH, ompA and blaPER-1 were investigated by polymerase chain reaction (PCR). It was determined that 60.3% (94/156) of all the isolates formed biofilm. Of these 94 isolates, 17 were weak, 33 were medium and 44 were strong. The mean biomass forming capacity of the isolates was found to be 2.23 ± 0.0033. Among the isolates included in the study (n= 156) the frequency of csgA, csuE, ompA, fimH and blaPER-1 genes were 71.2%, 32.1%, 21.8%, 7.1% and 3.2% respectively. The frequency of csgA, ompA, bap, csuE, fimH virulence genes were found to be 41.5%, 24.5%, 20.2% and 5.3% among biofilm positive isolates respectively. Biofilm-forming isolates were most commonly found in pulsotype II 19.1% (18/94), pulsotype IX 17.0% (16/94) and pulsotype VI 12.8% (12/94). In this study, when the distribution of virulence genes were compAred with the isolates that have weak, medium and strong biofilm, all of the studied genes were found to be more abundant in isolates with strong and medium positive biofilm production. This has shown that excluding fimH gene, csgA, csuE and ompA genes have contributed to the biofilm formation in invasive A.baumannii isolates, respectively.

Evaluation of therapeutic potential of intraperitoneal ozone gas in combination with insulin above cranial and spinal neuropathy in rats with diabetes mellitus.

OBJECTIVE: This study aimed to investigate the therapeutic effect of ozone in combination with insulin on cranial and spinal neuropathy in rats with diabetes mellitus (DM). MATERIALS AND METHODS: Sixty adult male Sprague Dawley rats were randomly divided into the following six groups (n = 10): control (C), ozone (O), diabetic (D), ozone-treated diabetic (DO), insulin-treated diabetic (DI), and ozone, insulin-treated diabetic (DOI). DM was induced by a single intraperitoneal (ip) streptozotocin injection (60 mg/kg), followed by 3 IU (ip) insulin administration for 60 days. Next, 1.1 mg/kg (50 µg/ml) ozone was administered to the O, DO, and DOI groups for 60 days. After inducing diabetes, the total oxidant status (TOS) and total antioxidant status (TAS) were measured; the oxidative stress index (OSI) was calculated. The brain and spinal cord tissues were obtained for histopathological evaluation. This cross sectional study was conducted in Dumlupinar University Laboratory Animals Research Center e.g 11.03.2015 ‒ 15.05.2015. RESULTS: TAS was higher in the DO, DI, and DOI groups than in the D group. TOS and OSI were lower in the DO, DI, and DOI groups than in the D group. Little pathological alterations with degenerated axons and vascular congestion were observed in the DO, DI, and DOI groups compared with the D group. CONCLUSION: Ozone with insulin can stimulate the endogenous antioxidant defense mechanism in diabetic neuropathy, thereby preventing reactive oxygen species-induced damage and protecting against cranial and spinal neuropathies (Fig. 6, Ref. 29).

The neuroprotective effects of 2-APB in rats with experimentally- -induced severe acute pancreatitis.

AIM: The objective of this study was to determine the neuroprotective effects of 2-aminoethyl diphenyl-borinate (2-APB) on the brains of rats with experimentally-induced severe acute pancreatitis. MATERIALS AND METHODS: Thirty Spraque-Dawley male rats with an average weight of 200-250 grams were randomly divided into three groups. Group 1: Sham group, Group 2: Severe acute pancreatitis group, Group 3: Treatment group with severe acute pancreatitis, given 2 mg/kg 2-APB before pancreatitis onset. In Groups 2 and 3, severe acute pancreatitis was induced by intraperitoneal administration of 1.5 g/kg L-arginine with a 1-hour interval. Tumor necrosis factor-α, interleukin 6, pancreatic amylase were all measured. Brain tissue samples were evaluated histopathologically. TUNEL staining method was used to visualize apoptotic cells. RESULTS: In Group 3, it was determined that the density of TUNEL-positive cells in the cerebral cortex has decreased, while the number of Bcl-2-positive cells had increased. In Group 3, it was observed that glial aggregation areas were diminished and histopathological changes were decreased as compared to Group 2. In Group 2, on the other hand, it was observed that in areas with glial cell aggregation, the density of TUNEL-positive glial cells had increased, while Bcl-2-positive cell reaction has been feeble. CONCLUSIONS: It was observed that 2-APB decreases neuronal apoptosis and glial cell aggregation (Tab. 2, Fig. 3, Ref. 21).

Effects of MMP-1 and MMP-3 gene polymorphisms on gene expression and protein level in lumbar disc herniation.

The aim of this study was to identify the possible correlation between polymorphisms in matrix metalloproteinase (MMP)-1 and MMP-3 and their corresponding protein levels in disc tissues obtained from patients with lumbar disc herniation (LDH) using biochemical and immunohistochemical analyses. Blood and disc samples were obtained from 100 patients with LDH who underwent a lumbar microdiscectomy. Based on the radiological degeneration, the patients were diagnosed with grade 2, 3, or 4 LDH. MMP-1 -1607 1G/2G and MMP-3 -1171 5A/6A were analyzed by real-time polymerase chain reaction. The expressions of MMP-1 and MMP- 3 were detected by biochemical and immunohistochemical analyses. We found no association between the MMP-1 polymorphism and disc degeneration and MMP-1 expression. However, patients expressing the 6A/6A and 5A/6A alleles of MMP-3 -11715A/6A showed higher MMP-3 expression, compared to those expressing the 5A/5A genotype. Additionally, the radiological degeneration grades were correlated with the histological degeneration scoring. Protein levels and immunopositive cell rates of MMP-1 and MMP-3 were associated with disc degeneration grades. Moreover, the MMP-1 and MMP-3 expression and the histological and radiological scores were positively correlated and the MMP-3 -11715A/6A polymorphism was associated with MMP-3 expression in herniated disc tissues. This study is the first to investigate polymorphisms in MMP-1 and MMP-3, as well as their corresponding protein expressions. We also quantified an association between the radiological degeneration grades and MMP-1 and MMP- 3 expression. Further genomic studies on MMPs could focus on the utilization of MMP-1 and MMP-3 as markers for the prevention and treatment of this disease.

Lack of association between the methylenetetrahydropholate reductase gene A1298C polymorphism and neural tube defects in a Turkish study group.

The etiology underlying neural tube defects (NTDs) is not fully understood and is believed to involve a complex milieu of genetic and environmental factors. The A1298C polymorphism in the methylenetetrahydropholate reductase gene (MTHFR) has been associated with mild risk for NTDs. In this study, the genotype distribution of the MTHFR gene A1298C polymorphism and the levels of serum homocysteine, vitamin B12, and folate were evaluated in 33 children with NTDs, their mothers, and 46 healthy controls. Genotyping of the A1298C polymorphism was performed by real-time polymerase chain reaction. The A and C allele frequencies in children with NTDs and their mothers were similar to controls (P = 0.160). The 1298AA and 1298CC genotype frequencies (P = 0.551 and 0.062, respectively) in children with NTDs and their mothers did not differ from controls. On the other hand, the 1298AC genotype frequencies in children with NTDs and their mothers were significantly different from controls (P = 0.025). The genotype frequency of 1298AC was lower in children with NTDs than in controls. There was no significant association between clinical distribution of NTDs and 1298AA/AC/CC genotypes (P > 0.05). Serum vitamin B12 levels were higher in children with NTDs than their mothers and controls (P = 0.001). There were no differences among serum homocysteine and folate levels in all groups (P = 0.494 and 0.141, respectively). Both genetic and nutritional factors are important in the etiology of NTDs. Thus, the A1298C polymorphism cannot be regarded as a major risk factor for NTDs.

Nimodipine can diminish oxidative stress in patients with severe head trauma.

AIM: Antioxidative effect of nimodipine was investigated in patient with severe head trauma. METHODS: The patients in group A were treated according to the standard procedures without nimodipine. Other patients in group B were treated with standard procedures plus intravenous nimodipine for a week. Three times per day, blood samples were taken from internal jugular venous saturation probe and central venous catheter for a week. The levels of malondialdehyde (MDA), reduced glutathione (GSH), nitrite, and nitrate, ascorbic acid, retinol and ß-carotene in the serum were measured. RESULTS: MDA levels in group B were significantly lower than those in the group A (P<0.05). As for GSH levels, it was observed that there was a significant increase in GSH levels in group B when compared to those in group A (P<0.01). Comparison of nitrate and nitrite levels in the serum of patient groups showed that these parameters were significantly higher in group B than those in group A (P<0.01). It was seen that there were a significant increase in ascorbic acid (P<0.01) and ß-carotene (P<0.05) levels in group B when compared to those in group A. Values of retinol levels were slightly higher in group B than that of group A, and there was no significant difference between the groups. CONCLUSION: Severe head trauma may increase oxidative stress. Administration of nimodipine may prevent the oxidative stress and may augment endogenous antioxidative defense systems in patients with severe head trauma.

Short aggrecan gene repetitive alleles associated with lumbar degenerative disc disease in Turkish patients.

We investigated a possible association between aggrecan gene polymorphism and lumbar degenerative disc disease in Turkish patients. One hundred 20-30-year-old patients with or without low back pain were selected for the study. Lumbar magnetic resonance imaging was performed on all patients. The patient group had low back pain clinically and degenerative disc disease radiographically. The control group included patients with and without low back pain: all were negative radiographically for degenerative disc disease. Genomic DNA was extracted from all participants. A PCR assay were used to evaluate variable number of tandem repeat polymorphism of aggrecan gene alleles to determine if there was any correlation with degenerative disc disease. Significant associations were found between short repeated alleles of the aggrecan gene and severe disc degeneration. A significant association was also found between short repeated alleles of the aggrecan gene and multilevel disc herniation as well as extrusion and sequestration types of disc herniation. In Turkish population, short repeated alleles of the aggrecan gene are associated with increased disc degeneration and disc herniation.

C677T polymorphism of the methylenetetrahydrofolate reductase gene does not affect folic acid, vitamin B12, and homocysteine serum levels in Turkish children with neural tube defects.

Association between neural tube defects (NTDs) and C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene was suspected, because the MTHFR gene codes for a key enzyme in folate metabolism. Its deficiency usually leads to significant reductions in plasma concentrations of folate, vitamin B(12) and methionine, whereas homocysteine levels are increased. We examined folate, vitamin B(12) and homocysteine serum concentrations and polymorphism of the C677T MTHFR gene in Turkish children with neural tube defects. Thirty-three children with NTDs, 26 mothers and 48 healthy individuals were studied. C677T MTHFR polymorphism was determined by melting curve analyses (LightCycler). The levels of folate, vitamin B(12) and homocysteine serum concentrations in NTDs were evaluated and compared, along with information concerning alleles of the MTHFR gene. C677T allele frequencies in NTD children and their mothers were similar to those found in controls. Serum folate and vitamin B(12) concentrations were significantly higher in NTD children than that of controls. Serum homocysteine concentrations were not significantly higher in NTD children and mothers. We concluded that C677T MTHFR gene polymorphism does not affect folic acid, vitamin B(12) and homocysteine metabolism in Turkish children with NTDs. C677T polymorphism of the MTHFR gene cannot be regarded as a major risk factor for NTDs in Turkish children.

Efficacy of nasopharyngeal culture in identification of pathogen in middle ear fluid in chronic otitis media with effusion.

PURPOSE: Chronic otitis media with effusion (OME) is the leading cause of hearing loss during childhood. In bacterial etiology of OME, the most frequent pathogens responsible are Haemophilus influenzae followed by Streptococcus pneumoniae and Moraxella catarrhalis. This study aimed at evaluating the accuracy of nasopharyngeal (NP) specimens in the identification of pathogens in the middle ear fluid (MEF) in patients with OME. MATERIALS AND METHODS: In this cross sectional, case-control study, 95 MEFs and 53 NP secretion specimens were obtained from 53 children. As a control group, 102 NP specimens were taken from children having an operation other than an otological disease. Conventional culture methods and multiplex-PCR method have been used to determine the etiology of OME; NP carriage between cases and control groups were compared using conventional culture methods. Pearson Chi-Square and Fisher's Exact tests were used in statistical analysis. RESULTS: Bacteria were isolated by culture in 37.9% of MEF specimens, 14.7% of which belonged to the group H. influenzae, S. pneumoniae and M. catarrhalis. PCR was positive in 30.5% specimens targeting the same pathogens. There was a two-fold increase in carriage rate of S. pneumoniae and H. influenzae in patients than controls for each pathogen. CONCLUSION: PCR is a more reliable method to detect middle ear pathogens in MEF in comparison with the conventional culture methods. The NP colonization wasn't found to be an indicator of the pathogen in MEF although middle ear pathogens colonize more in nasopharynx of diseased children.

A new visual field test in empty sella syndrome: rarebit perimetry.

PURPOSE: Several visual field defects can be seen in empty sella syndrome (ESS). In this study, the authors aimed to evaluate the visual field defects in patients with ESS by rarebit perimetry and to compare the results with Humphrey perimetry. METHODS: Left eyes of 13 patients with ESS and left eyes of 15 age-matched normal subjects were included in the study. Visual field testing was performed by Humphrey Visual Field Analyzer II (Fastpack 30-2 strategy) and rarebit perimetry (regular test). Statistical analysis was performed by independent-samples t-test, Mann-Whitney U test, receiver operating characteristic (ROC) curves, and Pearson correlation test. RESULTS: Humphrey perimetry mean deviation was -3.67 dB in control group and -6.06 dB in patients with ESS (p=0.12). Mean hit rate calculated by rarebit test was 91.8% in control group and 75.9% in cases with ESS (p=0.005). Area under ROC curve was 0.756 for Humphrey visual field test and 0.827 for rarebit hit rate (p=0.59). There was a significant correlation between rarebit hit rate and Humphrey visual field test mean deviation (r=0.755, p<0.001). CONCLUSIONS: Rarebit perimetry correlates significantly with Humphrey perimeter in detecting visual field defects related with ESS and has a higher sensitivity and specificity.

Genomic backgrounds of drug-resistant Streptococcus pneumoniae in Ankara, Turkey: Identification of emerging new clones.

Streptococcus pneumoniae exhibiting decreased susceptibility to penicillin are isolated with an increasing prevalence in Turkey during the last decade. This study was undertaken to investigate the molecular epidemiology of non-penicillin-susceptible pneumococci isolated in Ankara, Turkey. Among a population of 246 pneumococci, 90 pneumococci with penicillin MIC > or = 0.1 microg/ml were serotyped, genotyped by pulsed-field gel electrophoresis (PFGE), and sequence typed by multilocus sequence typing (MLST). The overall resistance to penicillin, cefotaxime, erythromycin, clindamycin, chloramphenicol, tetracycline, rifampicin, ciprofloxacin, and vancomycin were 36.6%, 4%, 27.6%, 10.9%, 5.3%, 22.4%, 4.5%, 2%, and 0, respectively. The most frequent serotypes were 14, 23B, 9V, 19F, 19A, and 23F. PFGE types represented 17 genetic clusters. PFGE and MLST data revealed that there were isolates identical or closely related to the Spain(9V)-3 ST 156 clone, Portugal(19F)- 21 ST 177 clone, and Spain(23F)-1 ST81 clone. Eleven serotype 14 isolates with emerging resistance to penicillin belonged to the ST 230 complex, a predominantly susceptible clone. Serotype 19A, 19F, and 7F variants of the ST 230 clone were also identified in the study population. Eight serotype 23B isolates with a new ST 1349 (18-13-8-6-3-6-8) created another clone with no relation to the currently defined international clones. Although the pandemic clones Spain(9V)-3, Portugal1(9F)-21, and Spain(23F)-1 are present in our region, the emergence of a new 23B clone with a unique ST and the emergence of resistance in the ST230 clone, has presumably contributed to the increase in the prevalence of drug-resistant pneumococci in Turkey.

The penetration of cefoperazone and sulbactam into the lumbar intervertebral discs.

Six patients received 1 g and six other patients received 2 g of cefoperazone and sulbactam 15 minutes before lumbar disc surgery. Liquid chromatographic analysis of disc tissue revealed that only patients receiving the 2-g dose had mean tissue levels above the minimum inhibitory concentration for Staphylococcus aureus and epidermidis.

Use of enhanced stimulation voltage to determine the severity of compressive peripheral nerve injury.

BACKGROUND: The aim of this study was to investigate whether enhanced stimulation voltage could be a predictor of the extent of injury in acute compressive peripheral nerve trauma. METHODS: The femoral nerves were exposed on both sides, in 11 anesthetized rabbits. Supramaximal stimulation voltage was used to produce a maximal-amplitude compound muscle action potential (CMAP) from the quadriceps femoris muscle. Afterward, the left femoral nerve was clipped for 1 minute, and the right femoral nerve for 5 minutes to produce an acute compressive injury. Immediately after removal of the clip, the proximal and distal sides of the clippage site were stimulated by gradually increased voltage until CMAPs were obtained. The same procedure was repeated at the 30th and 60th minutes. The ratio of the CMAP amplitudes obtained from proximal and distal stimulation was measured to establish a classification. RESULTS: The stimulation voltages and amplitudes of the CMAPs before clippage were similar with the after-clippage values obtained from distal stimulation (p > 0.05), but the after-clippage values obtained from proximal stimulation were different in both sides (p < 0.05). Doubled stimulation voltage was enough to obtain CMAPs on the left side, but eightfold the initial level was required on the right side. The amplitude ratios recovered to preinjury levels in all of the subjects on the left side, but only two showed recovery on the right side (p < 0.001). Histopathologically, there was axonal compression without discontinuity on the left side, whereas the fibers were dispersed on the right side. CONCLUSION: Stimulation voltage was found to discriminate the severity of the lesion in experimental peripheral nerve injury. Proximal to distal amplitude ratio seems to be a prognostic factor when the injury is less severe.