Aggressiveness and Patulin Production in Penicillium expansum Multidrug Resistant Strains with Different Expression Levels of MFS and ABC Transporters, in the Presence or Absence of Fludioxonil.

Penicillium expansum is the most common postharvest pathogen of apple fruit, causing blue mold disease. Due to the extensive use of fungicides, strains resistant to multiple chemical classes have been selected. A previous study by our group proposed that the overexpression of MFS (major facilitator superfamily) and ABC (ATP binding cassette) transporters constitute an alternative resistance mechanism in Multi Drug resistant (MDR) strains of this pathogen. This study was initiated to determine two main biological fitness parameters of MDR strains: aggressiveness against apple fruit and patulin production. In addition, the expression pattern of efflux transporters and hydroxylase-encoding genes that belong to the patulin biosynthesis pathway, in the presence or absence of fludioxonil and under in vitro and in vivo conditions were investigated. Results showed that the MDR strains produced higher concentrations of patulin but showed a lower pathogenicity compared to the wild-type isolates. Moreover, expression analysis of patC, patM and patH genes indicated that the higher expression levels do not correlate with the detected patulin concentration. The selection of MDR strains in P. expansum populations and the fact that they produce more patulin, constitutes a serious concern not only for successful disease control but also for human health. The above-mentioned data represent the first report of MDR in P. expansum associated with its patulin-production ability and the expression level of patulin biosynthesis pathway genes.

Synthesis and Characterization of Novel Copper Nanoparticles for the Control of Leaf Spot and Anthracnose Diseases of Olive.

Olive crop is frequently treated with copper fungicides to combat foliar and fruit diseases such as olive leaf spot caused by Fusicladium oleagineum and anthracnose caused by Colletotrichum spp. The replacement of copper-based products with more eco-friendly alternatives is a priority. Metal nanoparticles synthesized in several ways have recently revolutionized crop protection with applications against important crop pathogens. In this study, we present the development of four copper-based nanoparticles (CuNP Type 1 to 4) synthesized with a wet chemistry approach. The CuNPs were characterized using Transmission Electron Microscopy, Dynamic Light Scattering, Laser Doppler Electrophoresis, and Attenuated Total Reflection measurements. In addition, the activity of the four CuNP types was tested in vitro and in planta against F. oleagineum and Colletotrichum spp. In vitro sensitivity measurements showed that for both pathogens, mycelial growth was the most susceptible developmental stage to the tested compounds. Against both pathogens, CuNP Type 1 and Type 2 were found to be more active in reducing mycelial growth compared to the reference commercial compounds of copper oxide and copper hydroxide. In planta experiments showed that CuNP Type 3 and CuNP Type 4 exhibited a strong protectant activity against both F. oleagineum and Colletotrichum acutatum with control efficacy values significantly higher than those achieved by the applications of either reference product.

Bacillus subtilis MBI600 Promotes Growth of Tomato Plants and Induces Systemic Resistance Contributing to the Control of Soilborne Pathogens.

Bacillus subtilis MBI600 (Bs MBI600) is a recently commercialized plant-growth-promoting rhizobacterium (PGPR). In this study, we investigated the effects of Bs MBI600 on the growth of tomato and its biocontrol efficacy against three main soilborne tomato pathogens (Rhizoctonia solani, Pythium ultimum, and Fusarium oxysporum f.sp. radicis-lycopersici-Forl). Furthermore, the root colonization ability of the Bs MBI600 strain on tomato roots was analyzed in vivo with a yellow fluorescence protein (yfp)-labeled strain, revealing strong colonization ability, which was affected by the root growth substrate. The application of Bs MBI600 on tomato plants resulted in significant increases in shoot and root lengths. Transcriptional activation of two auxin-related genes (SiPin6 and SiLax4) was observed. Single applications of Bs MBI600 on inoculated tomato plants with pathogens revealed satisfactory control efficacy compared to chemical treatment. Transcriptomic analysis of defense-related genes used as markers of the salicylic acid (SA) signaling pathway (PR-1A and GLUA) or jasmonic acid/ethylene (JA/ET) signaling pathway (CHI3, LOXD, and PAL) showed increased transcription patterns in tomato plants treated with Bs MBI600 or Forl. These results indicate the biochemical and molecular mechanisms that are activated after the application of Bs MBI600 on tomato plants and suggest that induction of systemic resistance (ISR) occurred.

Apple Fruit Core Rot Agents in Greece and Control with Succinate Dehydrogenase Inhibitor Fungicides.

Core rot is a major postharvest disease of apple fruit that occurs worldwide and is caused by a complex of fungi. Despite the importance of the disease, little is known about its etiology in Greece. In this study, 325 fungal isolates obtained from fruit with core rot symptoms were identified to the species level using morphological characteristics and phylogenetic analysis. Fungal identification revealed that Alternaria alternata was the major disease agent (57.8% of the isolates), followed by Kalmusia variispora (27.8%), Botrytis cinerea (12%), and Fusarium spp. (3.3%). K. variispora is reported for the first time as an agent of core rot of apple and its pathogenicity was confirmed by artificial inoculation tests. In addition to disease etiology, field experiments were performed at two different orchards for 3 consecutive years (2017 to 2019). Experiments were conducted to determine the effectiveness of several classes of fungicides and the timing of application for control of the disease. Greater efficacy was achieved when fungicides were applied at the petal fall stage (flowers fading BBCH 67), while the most effective fungicides were the succinate dehydrogenase inhibitors fluxapyroxad, fluopyram, adepidyn, and penthiopyrad. The results of this study are expected to contribute to the optimization of disease management and reduce the yield losses caused by core rot pathogens in Greece.

Multidrug resistance of Penicillium expansum to fungicides: whole transcriptome analysis of MDR strains reveals overexpression of efflux transporter genes.

Penicillium expansum is the most common apple fruit postharvest spoilage agent that causes a disease known as Blue Mold. Disease control is based on fungicide use. However, development of resistance to fungicides hampers the success of this control method. Fungicide sensitivity monitoring studies in Greece revealed the presence of pathogen strains exhibiting simultaneous resistance to different chemically unrelated compounds (multidrug resistance, MDR). This study was initiated aiming primarily to test the hypothesis that the MDR phenotype is associated with overexpression of efflux transporter genes and to determine the fitness of the MDR isolates. The monitoring study (n = 264) and the measurements of sensitivity in terms of EC50 values to 9 different compounds revealed that almost 5% of the population was of the MDR type. In the selected MDR isolates, the highest resistant factors were calculated for fludioxonil and pyraclostrobin, while the same isolates were moderately resistant to cyprodinil, thiophanate methyl and fluxapyroxad. In the resistant strains no target site mutations were detected in the target genes of each fungicide class, while in addition, a synergistic activity was observed between fungicides and the drug transporter modulator verapamil in some isolates. To obtain a direct insight on the resistance mechanism, the transcriptome of 2 MDR and 1 sensitive isolates was sequenced using Illumina HiSeq 2500 and differences in efflux transporter gene expression profile were figured out. Gene expression profiling analysis was performed before and after the exposure of fungal mycelia to fludioxonil. This analysis revealed the up-regulation of several MFS transporter genes and a limited number of ABC transporter genes either before or after the exposure to fludioxonil in the MDR isolates. Expression results for genes with the highest expression levels were verified by qRT-PCR assays. Fitness components measurements revealed that MDR isolates were of lower mycelial growth and pathogenicity compared to sensitive strains but they were producing higher number of conidia. The above mentioned data represent the first report of MDR in P. expansum associated with overexpression of drug efflux transporters and contribute to our knowledge in the mechanisms associated with fungicide resistance development in this fungal species.

Ultrasound, Clinical, and Electrophysiological Findings in Persistent Carpal Tunnel Syndrome.

BACKGROUND AND PURPOSE: We present the clinical, electrophysiological, and nerve ultrasound findings in cases of persistent carpal tunnel syndrome (PCTS). METHODS: Eighteen PCTS patients underwent evaluation with the Boston Carpal Tunnel Syndrome Questionnaire (BCTSQ), electrophysiology, and nerve ultrasound with a mean of 3.5 months (SD ± 1.4) after open surgery. RESULTS: PCTS patients showed a mean symptom severity scale score of 3.1 (SD ± 1.1) and functional severity scale score of 3.2 (SD ± 0.9) in BCTSQ. Nerve conduction studies revealed axonal affection of the median nerve in 13/18 patients, ultrasound showed disturbed echogenicity in all patients, a pathological wrist to forearm ratio in 16/18 patients, and cross-sectional area enlargement of the median nerve at the distal wrist crease in 12/18 patients. Ultrasound documented scar tissue formation (in 12/18 patients), incomplete release of retinaculum flexorum (in 4/18 patients), and neuroma of the median nerve (in 2/18 patients) as PCTS cause. CONCLUSION: Our data show significant functional disability, axonal nerve damage, and scar tissue formation as common PCTS causes.

Clinical characteristics and management of a Greek female patient cohort with breast conserving treatment.

PURPOSE: The purpose of this retrospective single-center study was to examine histopathological characteristics and treatment options in a cohort of Greek female patients treated with breast conserving surgery (BCS) and to evaluate potential predictive factors of breast cancer (BC) local recurrence. METHODS: The clinic's medical records from 1995 up to the end of July 2016 were scanned in order to identify female patients treated with BCS. We recognized 1175 patients who underwent BCS, representing 35.8% of the entire sample (3281 patients). RESULTS: The mean age of the patients enrolled in this study was 54.7 years, with a median follow-up period of 58.5 months. Nine deaths (0.8%) were registered with 5-year overall survival (OS) rate being 100%. Regarding adjuvant therapies, radiotherapy (RT) was assigned to 94.4% of the patients, endocrine therapy to 86.0%, chemotherapy to 51.3%, while all therapies were given simultaneously to 38.3% of the sample patients. Herceptin was administered to 14.1% of the patients. Twenty-eight recurrences (2.4%) with 3 deaths (10.7%) were confirmed. Over all traditional parameters studied, only lymph node status appeared to be statistically correlated with local recurrence (p=0.005). T3 stage can be considered as a hint that this tumor size might be a risk factor of local recurrence. Age at diagnosis seems to be an independent factor of BC local recurrence. CONCLUSIONS: A very low rate of local recurrence (2.4%) was validated which can be attributed to patients being appropriately selected for BCS, who were then enrolled in this study, and to the uniformity of the RT plan.

Impact of Oncotype DX on chemotherapy assignment: a retrospective single-center study on female breast cancer patients.

PURPOSE: This study was designed to determine the Recurrence Score (RS) distribution and its associated risk assessments based on traditional clinicopathologic characteristics in a single-center breast cancer (BC) deriving cohort in Greece, and to evaluate the impact of the RS results on adjuvant treatment decisions applied in this cohort. METHODS: This was a retrospective, single-center study regarding Greek female patients with early-stage breast cancer (ESBC). From 2009 to 2015, 114 cases lacking unanimity in the multidisciplinary breast meeting (MDM) fulfilled the inclusion criteria. The RS of the Oncotype DX (ODX) assay was the main outcome. RESULTS: The mean RS in the sample was 16.38 (SD=6.87). RS was positively correlated with Ki-67 (p=0.008). A negative progesterone receptor (PR) was associated with a higher RS (p<0.05). RS was higher for cases of chemotherapy assignment (p<0.001). According to the oncologists' pre-DX assay recommendations, 62.8% of the patients would have been 'wrongly' assigned to chemotherapy, while 14.3% of patients would have not been recommended this treatment even though they should have. The overall chemotherapy recommendation was significantly altered after the ODX RS assay was carried out (p=0.008) and, in the sample, it diminished by 39.5%. CONCLUSIONS: The distribution of the ODX RS in the specific cohort of Greek women is similar to that reported in other geographic regions of the world. Knowledge of the RS resulted in a shift in treatment recommendations towards lower-intensity regimens and in a greatly reduced proportion of chemotherapy recommendations.

Prognostic Value of Nerve Ultrasound and Electrophysiological Findings in Saturday Night Palsy.

BACKGROUND AND PURPOSE: We report on the prognostic role of the cross-sectional area (CSA) enlargement and conduction block (CB) in radial neuropathy (Saturday night palsy [SNP]). METHODS: Reference CSA values were defined in 30 healthy subjects. Twenty-four patients with SNP underwent evaluation (Thessaloniki Hypesthesia Score [THS], Medical Research Council [MRC], ultrasound, electrophysiology). All patients were followed up 3 months after initial presentation. RESULTS: During initial evaluation, 13 patients showed pathological CSA (pCSA) and 11 normal CSA (nCSA). Fourteen patients showed conduction block (pCB) and 10 showed no conduction block (nCB). The site of lesion was recognized in 11 patients in the spiral groove, in 8 patients in the distal main trunk, just before the division to motor and sensory branch, while 5 patients showed a double site of lesion (spiral groove and supinators' canal). During follow-up, the pCSA group showed a mean MRC score of 2.8 (SD ± .7) and a THS of 1.8 (SD ± .4), while the nCSA group showed a mean MRC score of 4.1 (SD ± .9) and a THS of .7 (SD ± .3) (P < .001) (Table 4). On the other hand, the pCB group showed a mean MRC score of 3.7 (SD ± 1.1) and a THS of 2.7 (SD ± .9), and the nCB group showed a mean MRC score of 4.2 (SD ± 1.2) and a THS of 1.7 (SD ± .9) (P = .355, P = .013, respectively). CONCLUSIONS: The CSA enlargement, but not the CB, seems to have a negative prognostic role in patients with SNP.

Clinical characteristics and management of a Greek female patient cohort with breast ductal carcinoma in situ.

PURPOSE: The purpose of this retrospective single-centre study was to examine the histopathological characteristics of breast ductal carcinoma in situ (DCIS) lesions in a cohort of Greek female patients and describe our experience regarding the clinical management of the disease. METHODS: The medical records from 1995 up to mid-2014 were scanned in order to trace DCIS cases. One hundred and seventy two patients (6.8% of all breast cancer cases) were diagnosed with pure DCIS and no invasive components; 32.0% underwent a second surgery, mainly due to first surgery positive margins. RESULTS: Age at first surgery ranged from 27 to 79 years (mean±SD 50±11) and median tumor size was 10mm (interquartile range/IQR7equals;12mm). Comedo necrosis (CN) was identified in 28.5% of the cases. The detection of CN was significantly associated with older age at diagnosis, larger tumor size and lower probability of highly differentiated tumors. Radiotherapy (RT) and hormonotherapy (HT) were applied to 44.8% and 63.4% of the patients, respectively. CONCLUSIONS: We implemented international practices (surgery, radiotherapy and prophylactic hormonal therapy) to patients diagnosed with DCIS and have observed only two relapses. It is our belief that DCIS requires a multidisciplinary approach and patient-tailored therapy which can potentially contribute to minimization of the local recurrence risk.

Identification, characterization and mycotoxigenic ability of Alternaria spp. causing core rot of apple fruit in Greece.

Alternaria core rot is a major postharvest disease of apple fruit in several countries of the world, including Greece. The study was conducted aiming to identify the disease causal agents at species level, investigate the aggressiveness of Alternaria spp. isolates and the susceptibility of different apple varieties and determine the mycotoxigenic potential of Alternaria spp. isolates from apple fruit. Seventy-five Alternaria spp. isolates obtained from apple fruit showing core rot symptoms were identified as either Alternaria tenuissima or Alternaria arborescens at frequencies of 89.3 and 11.7%, respectively, based on the sequence of endopolygalacturonase (EndoPG) gene. Artificial inoculations of fruit of 4 different varieties (Fuji, Golden Delicious, Granny Smith and Red Delicious) and incubation at two different temperatures (2 and 25°C) showed that fruit of Fuji variety were the most susceptible and fruit of Golden Delicious the most resistant to both pathogens. In addition, the production of 3 mycotoxins, alternariol (AOH), alternariol monomethyl ether (AME) and tentoxin (TEN) was investigated in 30 isolates of both species. Mycotoxin determination was conducted both in vitro, on artificial nutrient medium and in vivo on artificially inoculated apple fruit, using a high performance liquid chromatography with diode array detector (HPLC-DAD). The results showed that most of the isolates of both species were able to produce all the 3 metabolites both in vivo and in vitro. On apple fruit A. tenuissima isolates produced more AOH than A. arborescens isolates, whereas the latter produced more TEN than the former. Such results indicate that Alternaria core rot represents a major threat of apple fruit production not only due to quantitative yield losses but also for qualitative deterioration of apple by-products.

The value of chemoprophylaxis against Enterococcus species in elective cholecystectomy: a randomized study of cefuroxime vs ampicillin-sulbactam.

HYPOTHESIS: Cephalosporins are widely used and considered to be effective as prophylaxis in biliary surgery. Nevertheless, they lack activity against enterococci. We conducted a study to compare the efficacy of ampicillin-sulbactam vs cefuroxime in preventing surgical site infections following elective cholecystectomy. DESIGN: A prospective randomized controlled trial. SETTING: A major tertiary care hospital. PATIENTS: Four hundred eighteen randomized patients (of 549 total), who from July 2002 to August 2004 underwent elective open or laparoscopic cholecystectomy with prospective assessment for development of surgical site infections for 1 month postoperatively. INTERVENTION: A single intravenous dose of 1.5 g of cefuroxime (group A, n = 207) or 3 g of ampicillin-sulbactam (group B, n = 211) was administered during induction of anesthesia. Bile and gallbladder mucosal cultures were taken intraoperatively from all patients. MAIN OUTCOME MEASURE: Number of postoperative surgical site infections. RESULTS: A postoperative surgical site infection was noted in 19 (4.5%) of 418 patients, 18 from group A and 1 from group B (P<.001). In the group that received cefuroxime, 15 (83.3%) of 18 surgical site infections were due to Enterococcus species. Intraoperative bactibilia as well as intraoperative gallbladder rupture were associated with surgical site infections (P<.001). CONCLUSIONS: A single dose of ampicillin-sulbactam favored better compared with cefuroxime for prevention of postoperative surgical site infections due to Enterococcus species after elective cholecystectomy. Ampicillin-sulbactam may be a better agent for antimicrobial prophylaxis in high-risk patients undergoing elective cholecystectomy, especially in a setting where the incidence of enterococcal infections is higher.