8^th Clinical Microbiology Conference October 26-28, 2017 Paris, France

Ana Banko is an associate Professor at the Institute for Microbiology and Immunology, Faculty of Medicine, University of Belgrade. She has completed her PhD at the same university, researching the potential correlations between genetic variability of EBV and different EBV-associated diseases. She is a researcher of the national scientific project: The Medical Significance of Biological Variability of Viruses. She is also a specialist in Microbiology with Parasitology, and a member of three national referent laboratories. She has published more than 30 publications.

Epstein-Barr virus (EBV) infection is associated with the broad range of malignancies. EBV nuclear antigen 1 (EBNA1) has the most notable expression in cells, and latent membrane protein 1 (LMP1) is the major EBV oncogene. The aims of this study were to characterize polymorphisms of EBNA1 and LMP1 genes in different EBV-related diseases and to investigate potential sequence patterns that correlate with the clinical presentations of these diseases. Plasma samples from patients with mononucleosis syndrome (IM) (128) and from patients after renal transplantation (T) (116), and 116 nasopharyngeal carcinoma (NPC) biopsies were tested in this study. LMP1 and EBNA1 genes were amplified and analyzed with sequence, phylogenetic and statistical evaluation. The study showed five newly discovered EBNA1 subvariants with noticeable clustering of P-thr-sv-5 in NPC isolates. LMP1 variability showed two newly discovered and NPC-specific variants: Srb1 and Srb2. The B95-8 and North Carolina variants revealed as possible predictors for favorable TNM stages. In contrast, deletions in LMP1 represented possible risk factors for the most disfavorable TNM stage. In addition, the number of specific 33-bp repeats of LMP1, less than reference 4.5, and the combinations of LMP1/EBNA1 variability (deleted LMP1/P-thr and non-deleted LMP1/P-ala), absolutely correlated with elevated levels of hepatic transaminases in IM patients. On the other hand, the LMP1/ EBNA1 polymorphism Med/P-thr was identified as a possible risk factor for TNM stage IVB or progression to the N3 stage in NPC patients. In conclusion, potential viral screening markers for European NPC and the primary EBV infection could be recognized.

Mahasin Wadi completed her PhD from AL Neelain University, Medical Microbiology, Faculty of Medical Laboratory Sciences in 2010 and completed her MSc from University of Khartoum in Medical Microbiology & Pharmacology, 1987. She worked at Dar ALUloom University, College of Medicine, Riyadh, Saudi Arabia in August 2014; Central Research Laboratory Khartoum, Sudan; Department of Clinical Laboratory Science, and Medical Microbiology at King Saud University, Saudi Arabia, Riyadh in 1988. She worked in bee honey as a natural antimicrobial product and has published several papers in reputed journals and participated in many international and national conferences. She issued a patent research about the antimicrobial activity of Sudanese bee honey. She has attended many workshops and seminars and was awarded certificate of prestigious author for the journal of Bacteriology & Parasitology 2011. She was awarded a medal on participating in workshop at King Saud University Saudi Arabia 2011. She is a member of many international associations: German Apitherapy Society, American Apitherapy Society, International Bee Research Association, European Society of Clinical Microbiology and Infectious Disease ESCMID and Sudanese Veterinary Association. She served as reviewer of various journals.

Objective: This study aims to investigate the effect of honey dressing on diabetic septic foot not responding to conventional dressing. 

Background: Honey is one of the most appreciated and valued natural products introduced to humankind since ancient times. The best characterized role of honey in wound healing is in the prevention and limitation of bacterial infection, thereby reducing the bioburden of the wound.

Method: Swabs were taken from wound surfaces of diabetic septic foot for identification, viable count determination of infecting organisms. The isolated organisms were subjected to sensitivity test for honey and some of the commonly used antibiotics. The wounds of diabetic patients were generously soaked with honey and the patients were given supportive treatment (Omdurman Teaching Hospital).

Result: The clinical isolate was sensitive to all tested honey samples. Daily application of honey on septic wounds of hospitalized patients, gave favorable results. These involved cleanliness of wounds, growth of healthy granulation tissue and prompt graft taking.

Conclusion: Use of honey against septic wound in hospitalized patient admitted for amputation above knee resulted in complete healing within one week. Honey seems to accelerate wound healing by early promotion of healthy granulation tissue. This attributed to its effectiveness in healing chronic infected wounds not responding to conventional antibiotics.

Oximes are small chemical compounds utilized as treatments for organophosphate toxicity. Organophosphorous nerve agents prevent the enzyme acetylcholinesterase from performing its function - breaking down the neurotransmitter, acetylcholine. Nerve agents inhibit acetylcholinesterase by phosphylation. Oximes dephosphylate acetylcholinesterase, restoring normal function. When this takes place, the end result is a potentially fatal by-product known as a phosphylated oxime. Phosphylated oximes may be dangerous because they can inhibit acetylcholinesterase more potently than organophosphates; resulting in toxicity rather than a cure. The objective of this study is to evaluate inhibitory capacity and the toxicity of phosphylated oximes

to mammalian cells. The series of experiments conducted involved varying amounts of different oximes (K027, 2-Pralidoxime, etc.) and organophosphates (asinphos, dicrotophos, etc.) on NIH-3T3 cells, and SH-SY5Y cells. These experiments included: in- cell westerns, colorimetric assays, flux analysis, mass spectrometry, and a novel on- cell western blot. These experiments will examine the contributions of oximes, organphosphates, and the combination of both chemicals on acetylcholinesterase function and off- target toxicity. The results of this study suggest that the combination of nerve agents and oximes increases toxicity within neuronal cells by allowing the ability to permeabilize the cell membrane and disrupt normal functioning. All experiments conducted are indicative that the treatment method of oximes is deadly, more so than nerve agents alone. From these results, it can be determined that oximes are not a safe and effective treatment method for organophosphate toxicity. The findings in this study have the potential to change how organophosphate intoxication is treated.

The human microbiome of multiple body sites is affected by critical illness and clinical interventions. When and how microbes change in intensive care unit (ICU) patients is not fully understood. This study investigated the short and long-term changes of microbiota composition in pharynx, feces, tracheobronchial and gastric secretion of 6 ICU patients. The association of clinical factors (medication, infections, mechanical ventilation) was studied for each case. The microbiota of clinical samples was phylogenetically characterized by 16S rRNA sequencing. All samples were divided into 3 groups (early, mid, late), depending on sampling day. Overall, the microbiome of both respiratory and gastrointestinal tract showed a clear loss of species-richness over the course of hospitalisation (seen by decreasing Chao1 indices). Low-diversity communities (one genus comprising over 75%) were detected in all patients, especially in pharyngeal, tracheobronchial and gastric samples, the majority after longer ICU stays. Microbial composition showed great interpatient differences, but Staphylococcus and Enterococcus were pathogens frequently observed in several patients. We found that patients with an infection showed the infecting pathogen in at least one sample before (3 of 5 patients) or after (all patients) the clinical diagnosis of the infection. In most of these cases, the same pathogen was detected simultaneously in multiple sample areas, suggesting colonization of different body habitats. Other clinical factors were not directly related to microbial changes in this study. Assessing the clinical influence of microbiota diversity-loss in relation to the abundance of nosocomial pathogens in critically ill patients is of great interest for the future.

BRCA1 is a multifunctional tumor suppressor, whose expression is activated by the estrogen (E2)-liganded ERα receptor. The activated ERα is a transcriptional factor which activates various genes either by direct binding to the DNA at E2-responsive elements (EREs) and indirectly associate with a range of alternative non-ERE elements. Interference with BRCA-1 expression and/or functions leads to high risk of breast or/and ovarian cancer. Previous study in our lab investigated the involvement of Human T-cell leukemia Virus Type 1 (HTLV-1) in breast cancer, since HTLV-1 Tax was found to strongly inhibit BRCA-1 expression. Another study revealed that long exposure of 12-O-tetradecanoylphorbol-13-acetate (TPA), which is one of the stress-inducing agents activated the HTLV-1 promoter. So here the involvement of TPA in breast cancer had been examined by testing the effect of TPA on BRCA-1 and ERE expression. The results showed that TPA activated both BRCA-1 and ERE expression. In the 12 hours TPA activated the two promoters more than others time, and after 24 hours the level of the two promoters was decreased. Tax inhibited BRCA-1 expression but failed to inhibit the effect of TPA. Then the activation of the two promoters was not through ERα pathway because TPA had no effect on ERα binding to the two promoters of the BRCA- 1 and ERE. Also, the activation was not via nuclear factor kappa B NF-κB pathway because when we added the inhibitory of NF-κB to the TPA, it still activated the two promoters. However, it seems that 53BP1 may be involved in TPA activation of these promoters because ectopic high expression of 53BP1 significantly reduced the TPA activity. In addition, in the presence of BI- the inhibitor of Protein Kinase C (PKC)- there was no activation for the two promoters, so the PKC is agonized BRCA-1 and ERE activation.

Rana Abdulrahim Alaeq is currently a PhD student in Department of Molecular Biology and Biotechnology, University of Sheffield, UK, under the supervision by Prof. (Hon. Cardiff) Milton Wainwright. In 2004, she awarded Master of Microbiology, Faculty of Science in Taibah University and Master of Medical Microbiology, Faculty of Biology, Medicine and Health in University of Manchester in 2013. She worked as a Teacher Assistant in Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, Taibah University and also cooperated in the educational laboratories of the Department of Medical Laboratory Technology like medical microbiology, medical parasitology and medical virology.

Metallic copper (Cu) surfaces have antimicrobial properties against a variety of different microorganisms and copper touch surfaces are likely to be increasingly used in public places including hospitals. Studies in the literature show that molecular mechanisms result in the rapid killing of Cu surface-exposed bacteria and yeasts result from a sharp shock of extreme and immediate Cu-ion overload combined with severe membrane and cell envelope damage, although similar low mutation rates have been observed in cells obtained from both Cu and control surfaces. The aim of this study was to determine the survival of bacteria on the surfaces of copper and plastic plumbing surfaces. The antibacterial activity of copper surfaces was determined by overlying suspensions of Staphylococcus aureus and Escherichia coli on copper and plastic surfaces. All pipes were sterilized and bacterial suspensions from colonies were prepared and then the pipes were contaminated by the bacterial suspension. The experiments were performed at 18-23°C and the results were assessed after a 20-day exposure. The numbers of viable bacteria in the suspension were determined by serial dilution and plating on Nutrient Agar plates; the plates being incubated at 37°C for 48 h. The results showed that low counts of Staphylococcus aureus were seen on copper surfaces, as compared with those obtained on the plastic, control surfaces, i.e., the results show that E. coli failed to survive on copper pipes. The number of bacteria isolated from the plastic surfaces was consistently higher than the number isolated from copper surfaces. The survival rate of bacteria on the copper surfaces was low and none of the inoculated bacteria survived after 20 days of exposure. Copper is well known to be an antibacterial, and its use in medical environments is likely to the lead to the continuous reduction of environmental microbial contamination, including MRSA. The studies presented here show that the incorporation of Cu in healthcare facilities may dramatically help reduce the environmental microbial burden and act as a useful adjunct to current infection prevention and control systems, despite the fact that bacteria will eventually acquire

resistance to the ion.

Background & Aim: H. pylori were found to be an important factor in the pathogenesis of peptic ulcer disease. The growing problem of antibiotic resistance by the organism in Pakistan thus demands the search for novel compounds; we screened the aqueous extracts of Camellia sinensis and also compared the anti-bacterial

Materials & Methods: All these biopsies were processed for detection of H. pylori by two rapid helicourease – indigenously developed rapid urease detection kits, culture and polymerase chain reaction (PCR).The 5% aqueous extract of fermented, semi fermented and non-fermented Green tea was prepared and their antibacterial potential was explored against 35 clinical isolates of H. pylori agar well diffusion technique. The Minimum Inhibitory Concentration (MIC) of the most susceptible tea products were also carried out by Micro-dilution method for the sake of comparison.

Results: Most of the screened H. pylori isolates were resistant to more than one of the antibiotics like metronidazole and clarithromycin. The most significant activity was obtained in non-fermented green tea with an average zone of inhibition along with MIC was around 32 mm (MIC 120-150 á»±g/ml), semi fermented product showed 28 mm (MIC 140-200 á»±g/ml) showed and fermented one showed 32 mm (MIC 120-200 á»±g/ml).

Conclusion: In conclusion, our results indicated that all processed forms of green tea extracts possessed some variable level of anti H. pylori activity. But non-fermented that is freshly plucked green tea leaves without any industrial treatment has profound effect with least MIC and thus considered as a suitable and safe candidate for the eradication of H. pylori particularly drug resistant species.

Denitrification is an ecological process that treats water to reduce the nitrate levels to acceptable values, both in nature and water treatment enclosures. In our study, soil samples were collected from agricultural lands with leguminous cultivation in the districts of North 24 Parganas and Hoogly of West Bengal, India. 63 samples from various agricultural felids were assessed for heterotrophic bacterial count along with their denitrifying properties. 80% of the samples were positive for Pseudomonas spp., confirmed by bacteriological and biochemical typing. 57% of the samples were positive for oxidase, catalase, citrate, mannitol and negative for indol, MR-VP, urease, glucose, lactose, and maltose suggesting the presence of Pseudomonas spp. 46% were other associated species which were Pseudomonas negative. Significant co relation was observed between soil quality parameters like TOC, phosphorus, DO, salinity and pH to the number of positive samples p≤0.05, co-relation coefficient R=0.82. The samples were pooled and molecular confirmation was done by 16SrRNA amplification showing bands at 1500bp, confirming the presence of Pseudomonas spp. The samples showed infections in the experimental zebra fishes significant at p≤0.01, and evolutionary analysis using MEGA6 confirmed the species as Pseudomonas otitidis. The pathogens having denitrifying properties were largely obtained from the Hoogly district followed by North 24 Parganas district, suggesting that the species are ubiquitous microflora of the system and they play an important role in the nitrification process. Degree of denitrification by the positive samples was significant at p≤0.05. Pseudomonas otitidis isolated from natural systems can be useful for industrial treatment of waste waters, with modification of the pathogenic trait, if cultivated on a large scale in the state of West Bengal. This study focuses on the positive quality traits of some animal pathogens, which needs to be studied

further and is the first report of its kind from these districts.

Cotton is a cash crop of Pakistan and Gossypium arboreum is a locally cultivated variety, which has considerable resistance against various biotic and abiotic stresses. This variety of cotton is considered as good reservoir of stress tolerance genes, while based on EST data mostly of its genes are uncharacterized. Universal stress protein-2 (USP-2) gene was identified in 15 days drought stressed leaves of G.arboreum-FDH-171. Full length of this gene was mutated at three different (M1usp-2, M2usp-2, M3usp-2) positions (fig: 1) in three separate clones in E. coli-uspABC-mutant and Pichia pastoris-gs115 strains for its functional validation under various abiotic stress treatments (NaCl 800 mM, PEG 8%, Heat 37-450C, Cold 40C). The expression of 1st mutant (M1usp-2) was noted as 8.3fold under NaCl stress and 9.7fold under PEG stress treatments, recombinant cells showed higher growth up to 10-5 dilution in spot assay as compared to control and other genes. The 2nd mutant form of USP-2 was expressed on induction but it was failed to initiate stress tolerant mechanism in both organisms. No significant difference was noted in between 3rd mutant form and wild type USP-2. However, all mutant forms showed little tolerance against heat and cold stresses. The results of this study showed that activity of USP-2 was enhanced in M1usp-2 by enhancing its ATP

binding capacity at 2X but wipe out in M2usp-2 with zero ATP-binding ability and 4X enhanced CMP capacity has no effect on activity of M3-usp-2. In silico analysis showed that 1st and 3rd mutant forms of USP-2 may directly involve in stress adaptive mechanism or it might be function as a signaling molecule to initiate stress mechanism.

Background: Since 1981, when the first AIDS case was reported, worldwide, more than 34 million people have been infected with HIV. Almost 95 percent of the people infected with HIV live in developing countries. As HBV & HIV share similar routes of transmission by sexual intercourse or drug use by parenteral injection, co-infection is common. Because of the limited access to healthcare & HIV treatment in developing countries, HIV-infected individuals are present late for care. Enumeration of CD4+ T cell count at the time of diagnosis has been useful to initiate the therapy in HIV infected individuals. The baseline CD4+ T cell count shows high immunological variability among patients.

Methods: This prospective study was done in the serology section of the Department of Microbiology over a period of one year from august 2012 to July 2013. A total of 13037 individuals subjected for HIV test were included in the study comprising of 4982 males & 8055 females. Blood sample was collected by vein puncture aseptically with standard operational procedure in clean & dry test-tube. All blood samples were screened for HIV as described by WHO algorithm by Immuno-chromatography rapid kits. Further confirmation was done by biokit ELISA method as per the manufacturer’s guidelines. After informed consent, HIV positive individuals were screened for HBsAg by Immuno-chromatography rapid kits (Hepacard). Further confirmation was done by biokit ELISA method as per the manufacturer’s guidelines. EDTA blood samples were collected from the HIV sero-positive individuals for baseline CD4+ T count. Then, CD4+ T cells count was determined by using FACS Calibur Flow Cytometer (BD).

Results: Among 13037 individuals screened for HIV, 104 (0.8%) were found to be infected comprising of 69(66.34%) males & 35 (33.65%) females. The study showed that the high infection was noted in housewives (28.7%), active age group (30.76%), rural area (56.7%) & in heterosexual route (80.9%) of transmission. Out of total HIV infected individuals, distribution of HBV co-infection was found to be 6(5.7%). All co-infected individuals were married, male, above the age of 25 years & heterosexual route of transmission. Baseline CD4+ T cell count of HIV infected patient was found higher (mean CD4+ T cell count; 283cells/cu.mm) than HBV coinfected patients (mean CD4+ T cell count; 91 cells/cu.mm). Majority (77.2%) of HIV infected & all coinfected

individuals were presented in our center late (CD4+ T cell count;< 350/cu. mm) for diagnosis and care. Majority of co- infected 4 (80%) were late presented with advanced AIDS stage (CD4+ count; <200/cu.mm).

Conclusions: The study showed a high percentage of HIV sero-positive & co-infected individuals. Baseline CD4+ T cell count of majority of HIV infected individuals was found to be low. Hence, more sustained and vigorous awareness campaigns & counseling still need to be done to promote early diagnosis and management.