Alicja Nowak-Zaleska, PhD, dissertation in the field of genetic differentiation of the genus Acinetobacter, a microbiologist, an assistant in the 1Department of Biology, Ecology and Sport Medicene. Roman Kotłowski, PhD, chemistry, an assistant in the Department of Molecular Biotechnology and Microbiology. Agnieszka Mikucka, MD, microbiologist, deputy head of Dept. of Microbiology, Nicolaus Copernicus University in Toruń, Collegium Medicum of L. Rydygier in Bydgoszcz. Eugenia Gospodarek, MD, microbiology, prof. NCU in Toruń, head of Dept. of of Microbiology Nicolaus Copernicus University in Toruń, Collegium Medicum of L. Rydygier in Bydgoszcz

The occurrence of multidrug resistant (MDR) strains of A. baumannii is a growing problem especially when concerning patients in intensive care units. One of the causes of antibiotic resistance is the presence of transporter proteins responsible for their removal. So far, for strains of the species A. baumannii, a system of transporting antibiotics composed of three proteins, AdeA, AdeB and AdeC responsible for resistance to gentamicin, ofloxacin, cefotaxime, and tetracycline has been described. Multiple alignment revealed a 39% similarity of CAP01997 protein of A. baumannii containing Pro-Ala repeats to EmrA that is part of the EmrA/EmrB/TolC transportation system of E. coli responsible for mutlidrag resistance. We took tryin to check whether there is a correlation between the number of Pro-Ala repeats in the EmrA homologue of A. baumannii and the resistance to antibiotics. In this study 79 MDR A. baumannii strains isolated from patients were analyzed. Resistance to antibiotics was determined on Mueller–Hinton agar plates using a Kirby–Bauer disk diffusion test. The number of CCTGCA repeats encoding Pro-Ala repeats in EmrA-homologue of A. baumannii was determined using PCR method and capillary electrophoresis. The results of this research showed significant correlation between resistance to tobramycin and netilmicin, susceptibility to imipenem and the numbers of repeated Pro-Ala sequences in the CAP01997 protein, a homologue of EmrA. Based on presented results (p<0.0001) we can conclude that EmrA-homologue of A. baumannii confers resistance to Imipenem and Netilmicin depending on the presence of 7 and 5 Pro-Ala repeats in CAP01997 protein, respectively.

Persia D. Dadgardoust graduated with a Bachelor of Science degree in Medical Technology in 2007 from the University of Santo Tomas and is likewise a registered Medical Technologist in her country, the Philippines. She graduated from Medicine from the same institution in 2011 and is currently an Otorhinolaryngology – Head and Neck Surgery resident at the University of Santo Tomas Hospital. Her interests include Otology/Neuro-Otology, Audiology and Head and Neck diseases.

This case is set at a clinical division of a tertiary hospital and aims to present a case of tuberculosis of the thyroid gland in a female who presented with a left lateral neck mass, to discuss the incidence of tuberculosis of the thyroid gland in patients with the same profile as our patient and to discuss the diagnostic modalities and treatment for patients with the said disease. Patient is a 55-year old female who presented with a 1 year history of a gradually enlarging left lateral neck mass accompanied by cough, hoarseness, weight loss and intermittent fever. Tuberculosis work-up was negative and was ruled out. Nasal endoscopy revealed nasopharyngeal mass. Ultrasound of the neck was consistent with an evolving follicular neoplasm and fine needle aspiration biopsy was inconclusive. Due to risk factors in the patient, total thyroidectomy with bilateral selective neck dissection and nasopharyngeal biopsy were done. Final histopathology results were consistent with a tuberculous process. Patient was then started on anti-Koch’s regimen. Few have been available in current literature regarding the incidence of tuberculosis in the thyroid gland. According to these reports, diagnosis was often made post-operatively or postmortem. Some advocate the use of ancillary procedures such as ultrasound and CT scan to aid in the diagnosis. Repeated aspirations with bacteriological studies and staining methods can be recommended to avert a more radical management. A course of anti-Koch’s regimen would suffice to treat this disease masquerader.

Objective: Because bloodstream infections have a high mortality rate, rapid microbiological diagnosis is required to clinical efficient therapy. To develop a method of fast bacteria identification for pathogens causing bloodstream infection by GeXP analysis system based on the bacterial highly conserved region of 16S rDNA gene. Methods: We performed culture and real-time 16S rDNA gene PCR followed by direct sequencing. Clinical amples were cultured on Columbia blood agar plates were incubated aerobically for 48 h at 37°C and Brucella blood agar plates were incubated under anaerobic conditions for 72 h at 37°C. The 16S rDNA gene were amplified using the common methodology and sequenced with primer 5′-GAGCGGATAACAATTTCACACAGG-3′. Results: Ten clinical frequent bacterial strains were detected correctly at the species level . Candida albicans, herpes simplex virusⅠ、Ⅱ, HBV DNA and the blank control were all negative. The identification concordance was 100% by GeXP analysis system and conventional culture method at the genus level and it was 87.8% at the species level（36/38）. Gram-positive and Gram-negative bacilli bacteria accounted for 48. 9% and 43. 8% respectively , and fungi for 7. 3% in 252 strains isolated from blood specimens. The most frequent Gram-negative bacilli isolated were Escherichia coli of 66 strains accouting for 26.2% , Salmonella of 46 strains accouting for 18.3% , Klebsilla pneumoniae of 34 strains accouting for 13.5% . The most frequent gram-positive pathogenic bacteria isolated were Staphylococcus aureus of 31 strains accouting for 12.3% and coagulase negative Staphylococcus coagulase of 25 strains accouting for 9.9%. Conclusion: 16S rDNA Gene PCR with a GeXP genetic analysis system could detect blood culture positive sample directly and it was a fast, accurate mehod of bacteria identification for pathogens causing bloodstream infection.

Today, infectious diseases present new challenges and new potential for epidemics. Timely recognition of emerging infections requires early warning systems to detect such problems so that they may be promptly investigated and controlled before they evolve into public health crises. In addition, professional expertise, laboratory support and research capabilities are required to address the changing threats from emerging infections. The Centers for Disease Control and Prevention are on the front lines of the war on disease. CDC disease detectives battle the world's gravest health threats as they occur throughout the world. Each year, the CDC's laboratories receive thousands of specimens- blood, tissue containing unidentified disease-causing microbes in search of a diagnosis. Some are so deadly that scientists wear contamination-proof in order to investigate these killer microbes. As with many issues of health, education is a vital part of the battle against the spread of infectious diseases. By learning what threats are posed by emerging infectious diseases and by changing our behavior we can reduce the risk. With all the hi-tech approaches used in combating infectious disease causing microbes, it is important to remember that each individual can play a critical role in preventing and controlling infection. While a healthy immune system is the best defense, basic hygiene, such as proper hand-washing. and keeping the kitchen and bathroom clean, can help defend against harmful microorganisms.

Andrews Obeng Affum from Nuclear Chemistry and Environmental Research Centre, National Nuclear Research Institute, Ghana

In recent times, surface water resource in the Western Region of Ghana has been found to be inadequate in supply and polluted by various anthropogenic activities. As a result of the above problem, the demand for groundwater by the human populations in the peri-urban communities for domestic, municipal and irrigation purposes has increased without prior knowledge of its water quality. Water samples were collected from 14 public hand-dug well’s during the rainy season in 2013 and investigated for total coliforms, E.coli, mercury (Hg), arsenic (As), cadmium (Cd) and physico-chemical parameters. Multivariate statistical analysis of the dataset and a linear stoichiometric plot of major ions were applied to group the water samples and to identify the main factors and sources of contamination. Hierarchal cluster analysis revealed 4 clusters from the hydrochemical variables (R-mode) and 3 clusters in the case of water samples (Q-mode) after z-score standardization. Principal component analysis after a varimax rotation of the dataset indicated that 4 factors extracted explained 93.3% of the total variance, which highlighted salinity, toxic elements and hardness pollution as the dominant factors affecting groundwater quality. Cation-exchange, mineral dissolution and silicate weathering influenced groundwater quality. The ranking order of major ions were Na+>Ca2+>K+> Mg2+ and Cl-->SO42->HCO3-. Based on Piper plot and hydrogeology of the study area, sodium chloride (86%), sodium hydrogen carbonate and sodium carbonate (14%) water types were identified. Although Escherichia coli were absent in the water samples, 36 % of the wells contained total coliforms (enterobacter species) which exceeded the WHO guidelines limit of 0CFU/100ml water for microbes in drinking water. With the exception of Hg, the concentration of As and Cd in 79 % and 43 % of the water samples exceeded the WHO guideline limits of 10 μg/L and 3 μg/L for drinking water respectively. Reported values in some areas in Nigeria, Malaysia and United States of America, indicated that the maximum concentration of Cd was low and As high in this study. Health risk assessment of Cd, As and Hg based on average daily dose, hazard quotient and cancer risk were calculated. To conclude, multiple natural processes and anthropogenic activities from non-point sources contributed significantly to groundwater salinization, hardness, toxic element and microbiological contamination of the study area. The outcome of this study can be used as a baseline data to prioritize areas for future sustainable development of public wells.

Borsa BA from Istanbul Kemerburgaz University School of Medicine, Department of Medical Microbiology, Istanbul

Objectives: Brucellosis is difficult to diagnose based on clinical symptoms. Therefore, the diagnosis mostly relies on the results of serological testing. ODAK Brucella Coombs Gel Test (CGT) is a novel and rapid gel microcolumn agglutination test which is performed in microcolumns containing gel matrix and Coombs antibodies. In this study, we aim at comparing CGT with other commonly used serological tests. Methods: In this study, we included totally 150 blood samples of patients preliminary diagnosed as Brucellosis in Igdir which is an endemic area in Turkey. 50 of these were selected out of Rose Bengal (RB) and Standart Tube Agglutination (STA) test (+) samples, other 50 out of RB and STA (-) samples, and the last 50 out of RB (+) and STA (-) samples. We performed CGT and Brucellacapt for all samples. Coombs Wright test is performed for only STA negatives. 1/160 and higher titers were accepted as positive result for all methods except for RB. Results: Although all of the RB (-) STA (-) negative samples were also found negative with Coombs Wright, 2 samples (4%) with CGT and 5 more samples (7 in total, 14%) with Brucellacapt were found positive. Among 50 RB (+) STA (-) samples, 18 (36%) were found positive with Coombs Wright test, 28 (46%) were positive with CGT and 37 (74%) were positive with Brucellacapt. All 50 RB (+) STA (+) samples were also positive with CGT and Brucellacapt except for one sample which was found negative with CGT. Besides, results of 14 (28%) and 16 (32%) samples were found higher positive at least one titer with CGT and Brucellacapt respectively. However, Brucellacapt results of three patients were one titer less than in GT and STA. Among all RB positives, 87 (87%) samples were found positive with Brucellacapt while 77 (77%) samples were positive with CGT. Moreover, among all the RB negatives, 48 (96%) samples were found negative with CGT and 43 (86%) samples were positive with Brucellacapt. Conclusion: Currently, CGT is the only rapid (<1 hours) serological test which Coombs antibodies are used. Our results showed that negative results of RB were not found reliable enough as a screening test compared to GCT. However, positive RBT results confirmed with STA were almost always, in most of the cases with higher titers, positive with CGT and Brucellacapt. On the other hand, even if STA is found negative with RB positivity, samples still must be investigated with Coombs Wright, CGT or Brucellacapt. Consequently, CGT may be used as a rapid screening test instead of RB and furthermore, it has similar sensitivity with the other confirmation tests which Coombs antibodies are used. Therefore, ODAK Brucella Coombs Gel Test seems to be a very useful diagnostic tool for Brucellosis.

Ying Tang obtained an MSc in preventive veterinary medicine from Sichuan Agriculture University, P.R. China in 2012. My research program in MSc was the study on construction and immunogenicity of PEDV/ TGEV DNA vaccine, which was completed in Key Laboratory of Animal Disease and Human Health of Sichuan Province, China. During my master’s degree I have also completed another work in isolation and identification of Japanese B encephalitis virus, which was part of a research program on pathogen and hazard risk of zoonoses in earthquake affected area in Sichuan.

Salmonella enterica infection affects a wide range of animals and human. The avian specific serotype S. Pullorum infection produces systemic disease followed by a carrier state in convalescence birds. In comparison with S. Enteritidis, S. Pullorum has been found to induce increased levels of IL-4 and lower levels of IFN-γ in the spleen of infected birds which may switch the immunity from a pro-inflammatory to an anti-inflammatory response. Different immune responses between persistent Salmonella (S. Pullorum) and non-persistent (S. Enteritidis and S. Gallinarum) serotypes were compared ex vivo using Salmonella-infected macrophages and CD4+T lymphocytes. In comparison with S. Enteritidis and S. Gallinarum which induced an IFN-γ-producing Th17 response, macrophages infected with S. Pullorum had a reduced expression of IL-18 and IL-12α and stimulated proliferation of Th2 lymphocytes with reduced IFN-γ and increased IL-4. However, our data shows no evidence of clonal energy or immune suppression induced by S. Pullorum. In conclusion, S. Pullorum modulates host immunity from a dominant IFN-γ-producing Th17 response towards a Th2 response which may promote the persistent infection in chickens.

I-Hsiu Huang is an Assistant Professor at Department of Microbiology and Immunology Medical College, National Cheng Kung University, Taiwan

Clostridium difficile is a gram positive, spore forming obligate anaerobic bacteria and is the leading cause of the nosocomial antibiotic associated diarrhea. C. difficile infection (CDI) results in diarrhea and severe pseudo membrane colitiss (PMC) and toxic mega-colon. Before pathogens can cause disease it needs to attach to the host cell first. Therefore, our studies focus on non-toxin virulence factors responsible for C. difficile colonization. By bioinformatics analysis, orf CD2831 was predicted to encode for a large collagen-binding protein on the surface of C. difficile and was renamed as Cell surface protein 1 (Csp1). ELISA studies demonstrated that recombinant Csp1 binds specifically to collagen Type I. Previous in vitro studies has revealed that the metalloprotease CD2830 cleaves Csp1. Cyclic diguanylate (3’, 5’cyclic diguanylic acid) (c-di-GMP), a secondary messenger regulates the virulence and biofilm formation in many bacterial pathogens. Transcriptional analysis has indicated that c-di-GMP can increase the transcriptional level of Csp1 while down regulate CD2830 through riboswitches. The aim of our study then was to elucidate the relationship between c-di-GMP, CD2830 and Ccsp1 in C. difficile. In our preliminary data, we used cell fractionation and Western blot to analyze the localization of Csp1. Csp1 was found mostly secreted into the culture supernatant rather than on the cell wall under laboratory condition. Inactivation of CD2830 shifted the localization profile of Csp1 toward cell wall anchoring suggesting that Csp1 is secreted via cleavage by CD2830. Next, artificial induction of the cytoplasmic c-di-GMP level in C. difficile resulted in overexpression and prominent cell wall localization of Csp1. In summary, we have identified a novel collagen-binding surface protein in C. difficile that is bi-regulated by a metalloprotease and the secondary messenger c-di-GMP. Future works will focus on unraveling the role of Csp1 in C. difficile pathogenesis using animal studies.

Background: Associated pneumonia with antimicrobial resistant pathogens is a worldwide concern. New antimicrobial agents are sparse and such infections will become a major challenge. Liposomal formulations are of proven superiority in delivering drugs to end organs and have the ability to protect drugs from metabolites and other host's harsh materials and at the same time reduce toxicity. The goal of this study is to evaluate different neutral and negative liposomal-gentamicin formulations for their encapsulation efficiency, stability, and antibacterial activities. Method: Neutral liposomal-gentamicin (NLG), negative charged liposomal-gentamicin-1 (NELG-1) & negative charged liposomal-gentamicin-2 (NELG-2) were prepared by dehydration-rehydration method. The particles sizes in the formulations were measured by light scattering technique. Loaded gentamicin within different liposomal nanoparticles was measured by microbiological assay. Minimum inhibitory concentration (MIC), minimum bactericidal concentrations (MBC), and killing time curve were studied on several bacterial gram negative / positive strains. Liposomal formulations’ stability were studied within different biological conditions include plasma, sputum, and BAL at 37 ᵒC. Results: The encapsulation efficiency of gentamicin within NLG, NELG-1, and NELG-2 were 1.8%, 37.2% and .43.6% respectively. Formulations stability of each of NLG, NELG-1, and NELG-2 in presence of some of the biological fluids such as bronchial alveolar lavage (BAL), human plasma, and sputum were studied at 37ᵒC for 48 hours. NLG, NELG-1, and NELG-2 were kept interestingly amount of gentamicin among those biological fluids, but when exposed to human plasma their content of gentamicin were mild dropped down. Bacterial eradication of several planktonic pathogenic ATCC strains by NLG was 2-fold lower than free gentamicin, whereas NELG-1 and NELG-2 eradicated them as same as free gentamicin. The killing time curve values at 1, 2 and 4 time MIC for our formulations against P. aeruginosa were better than free gentamicin. Interestingly, NELG-2 reduced biofilm formation by 2-fold on day 2 better than NLG and NELG-1 compared to free gentamicin. Conclusion: All formulations exhibited a bactericidal effect able to eradicate bacterial strains at low concentration when compared to free gentamicin. On the other hand, these formulations exhibited improved killing time and anti-biofilm forming. Our endeavors are ongoing to examine theses formulations toxicity profile.

Clostridium difficile is a Gram-positive anaerobic human pathogen capable of withstanding multiple antibiotics. C. difficile infections (CDIs) is the leading cause of antibiotic-associated diarrhea worldwide. C. difficile exerts its action through the production of two glycosylating toxins, Toxin A and Toxin B. Similar to many other pathogens, C. difficile has the ability to bind and colonize the intestinal epithelium, however little is known about cell surface proteins that contributes to its adherence. Genome analysis of multiple C. difficile strains revealed a putative Type IV pilus gene cluster. Type IV pilus (TFP) is a unique bacterial appendage that has been reported in many Gram-negative bacteria to play an important role in motility, adherence, and biofilm formation. In our study, we aim to characterize the role of TFP in C. difficile pathogenesis. Results indicated that the TFP gene cluster was expressed constitutively in two toxin-encoding strain of C. difficile. To further investigate the functionality of TFP in C. difficile, insertional mutations were made in pilA, which encodes for putative pilin subunits of TFP, and in pilT, which encodes for a putative retraction ATPase associated with TFP disassembly. Interestingly, unlike previously reported in Clostridium perfringens, TFP in C. difficile did not appear to be involved in twitching motility. We further demonstrated that TFP was expressed on the C. difficile surface and was involved in biofilm formation. Unexpectedly, preliminary animal studies indicated that C. difficile mutants lacking TFP was more virulent.. We anticipate this study will reveal the function of TFP in C. difficile and provide alternative target for developing a rapid C. difficile detection method in the future.

Rita Berisio is Senior Researcher at the Insitute of Biostructures and Bioimaging of the “The National Research Council”. She has been responsible for a number of funding initiatives for example Coordinator of a project funded by the Mizutani Foundation for Glycoscience, grant-holder of the COST Action BM1003, Coordinator of a project of the Indigo Linking Programme, funded by EU. Her activities focus on structure/function relationship in macromolecules of biological interest combining crystallography and other physical-chemical techniques with molecular biology and biochemical studies. She is the co-author of about 95 publications on international journals among which Nat StructBiol, Mol Cell, JACS and PNAS.

There is an overall consensus that to develop improved drugs and vaccine control strategies, we need to gain a better understanding of the biology of involved pathogens. Innate immune recognition of bacterial pathogens is based on the detection of constitutive and conserved products of microbial metabolism on the bacterial cell surface. Lipopolysaccharides, lipoproteins, peptidoglycan and lipoteichoic acids are all unique molecules synthesized by bacteria but not eukaryotic cells. These products denominated Pathogen Associated Molecular Patterns are considered as the key molecular signatures of microbial invaders by the innate immune system and their recognition signals the presence of infection. The molecular arsenal of human host immune responses and small molecule mediators together define a dynamic host-pathogen interactome. Therefore, targeting proteins involved in host-pathogen interactions is a promising strategy for the development of new and effective anti-microbial drugs. Structural biology including molecular biology, biochemistry and biophysics concerned with the three-dimensional structure of biological macromolecules is of great interest to biologists since macromolecules carry out most of the cellular functions. As such, it has enormous impact on biomedical research and on biology in general. A tour through applications of structural biology to the understanding of molecular determinants of cellular processes involved in bacterial diseases will be presented.

Boutheina Ben Abdelmoumen Mardassi is a Doctorate in Veterinary Medicine from National School of Veterinary Medicine at Sidi Thabet, Tunis-Tunisia. She has completed her PhD from Biotechnology Research Institute at Montreal (BRI) and Montreal University in Canada. She was Post Doctorate from Armand-Frappier Institute at Montreal, Quebec, Canada. Since 2000, she is a Permanent Researcher and a Head of Mycoplasmas Laboratory at Pasteur Institute of Tunis. She has published more than 15 papers in avian and human scientific journals.

Mycoplasma synoviae is an important pathogen of both chickens and turkeys which causes synovitis, airsacculitis and egg shell apex abnormalities in chickens and may result in significant economic losses in the poultry industry. Mycoplasma synoviae has a great capacity to change its surface exposed antigens. Two major immunodominant and surface exposed membrane proteins, MSPB and MSPA were proved to be antigenically variable and considerable variation in their size and expression was reported within and among several strains of M. synoviae. This antigenic variation may contribute to the inefficiency of sero diagnostic tools and therefore jeopardize control and antibiotic therapy programs. In this study we sought to analyze the antigenic variability and its association with the antibiotic susceptibility of 23 Tunisian field isolates of Mycoplasma synoviae. Antigenic variability was determined by Western blot and colony blotting using anti-sera directed against MSPA and MSPB domains. All field strains were antigenically homogenous with the same antigenic pattern but significantly different from that of M. synoviae reference strain. Enrofloxacin, doxycyclin, oxytetracyclin and aivlosin are among the most widely antibiotic families used in poultry in Tunisia. In vitro susceptibility of the 23 Mycoplasma synoviae field isolates for these antibiotics was determined by the micro broth dilution method. Resistance to enrofloxacin with MIC ≥32 µg/ml was demonstrated forall isolates while a decrease of susceptibility to aivlosin for 50% of isolates was observed. This study argues for the predominance in Tunisia of a single serotype of M. synoviae that is fully resistant to enrofloxacin and less sensitive to aivlosin.

Boutheina Ben Abdelmoumen Mardassi is a Doctorate in Veterinary Medicine from National School of Veterinary Medicine at Sidi Thabet, Tunis-Tunisia. She has completed her PhD from Biotechnology Research Institute at Montreal (BRI) and Montreal University in Canada. She was Post Doctorate from Armand-Frappier Institute at Montreal, Quebec, Canada. Since 2000, she is a Permanent Researcher and a Head of Mycoplasmas Laboratory at Pasteur Institute of Tunis. She has published more than 15 papers in avian and human scientific journals.

The nuclease activity of mycoplasmas is an important factor in their pathogenicity. Nucleases may produce a broad spectrum of biological effects in hosts. In the present study, the ability of Mycoplasma meleagridis for nuclease synthesis was reported. Nuclease activity was found to be associated with a membrane protein named Mm19. BLASTP search analysis revealed a significant sequence similarity with a type II restriction enzyme belonging to the RE_AlwI super family. Further, Bio-informational analysis allowed the identification of a DNA methyl transferase which together with restriction enzymes forms restriction-modification (R-M) systems offering bacteria an effective defense system against foreign DNAs. Two sequences encoding for an endo nuclease of the RE_AlwI super family and for its DNA methyl transferaseare found juxstaposed and oppositely oriented in Mycoplasma meleagridis genome sequence. Like most of type II restriction enzymes, Mm19-associated nuclease activity was enhanced with Mg2+. Mm19 showed significant homologies with AlwI related sequences in other mycoplasmas (Mycoplasma mycoides subsp. capri GM12 and Mycoplasma bovis PG45) and other bacterial species such as Streptococcus, Bacillus and Staphylococcus. Based on sequence alignments, the 714-aa methylase sequence was found to harbor two putative conserved domains differently organized compared to other mycoplasmas methylases domains. In fact, these two putative conserved domains were found to be located in the same CDS in Mycoplasma meleagridis methylase while they were disjointed in two separated CDSs in other mycoplasmas methylases. The Mycoplasma meleagridis Mm19 encoded-product might be involved in pathogenicity and in the survival of this mycoplasma in the host.

Helmi Mardassi after obtaining his Doctorate in Veterinary Medicine (1988) at the Tunisian National School of Veterinary Medicine, he moved to the University of Montreal (Canada) where he completed a Master degree in Microbiology and Animal Pathology. In 1996, he obtained his PhD degree in Molecular Virology at the Institut Armand-Frappier (University of Quebec, Canada) and then joined the Biotechnology Research Institute of Montreal for a Post Doctoral Training. Actually he is leading a research unit focusing on molecular epidemiology, evolution and genetics of Mycobacterium tuberculosis. He has published more than 26 papers in reputed journals.

The IS6110 insertion sequence, a member of the IS3 family of insertion sequences was found to be specific to the Mycobacterium tuberculosis complex (MTBC) and contributes significantly to its pathogenicity. Here we explored the evolution of the IS6110 sequence by analysing the genetic variability and the selective forces acting on its transposase-encoding open reading frames (ORFs) A and B (orfA and orfB). For this purpose, we used a strain collection consisting of smooth tubercle bacilli (STB), an early branching lineage of the MTBC and present day M. tuberculosis strains representing the full breadth of genetic diversity in Tunisia. In each ORF, we found a major haplotype that dominated over a flat distribution of rare descendent haplotypes consisting mainly of single- and double-nucleotide variant singletons. The predominant haplotypes consisted of both ancestral and present-day strains suggesting that IS6110 acquisition predated the emergence of the MTBC. There was no evidence of recombination and both ORFs were subjected to strict purifying selection as demonstrated by their dN/dS ratios and Tajima’s D statistics. Strikingly, the purifying selection acting on orfA proved much more stringent suggesting its critical role in regulating the transpositional process. Maximum likelihood analyses further excluded any possibility of positive selection acting on single amino acid residues. Taken together our data fit with an evolutionary scenario according to which the observed variability pattern of the IS6110 transposase-encoding ORFs is generated mainly through random point mutations that accrued on a functionally optimal IS6110 copy, whose acquisition predated the emergence of the MTBC complex.

Evelien Kieckens worked in a tissue engineering lab during her internship’s Master thesis on the development of a bioactive meniscus using polymeric scaffold and hydrogels. She obtained the degree of Master in Biomedical science in 2011 at Ghent University. In 2012, she started her PhD at the faculty of Veterinary Medicine at Ghent University, where she has been working on the clearance of EHEC infections in ruminants. Until now she is first author of one publication in Applied and Environmental Microbiology and first author and co-author of several manuscripts in preparation.

Enterohemorrhagic Escherichia coli (EHEC), like E. coli O157:H7 are frequently detected in slaughterhouses, as cattle are the main natural reservoir of EHEC showing no symptoms upon infection. However, for humans the consequences can be severe, leading to hemorrhagic colitis and hemolytic uremic syndrome. An EHEC infection can persist in herds for longer periods. The immune response against EHEC cannot clear the infection rapidly as infected animals can secrete the bacteria during several months. We observed that a first infection elicits a primary immune response, which was unable to protect from an experimental re-infection. Furthermore, the duration of shedding of the first infection was shorter (<14 days) compared to the duration after a re-infection (>28 days). We hypothesized that these results may reflect a suppression of certain pathways of the immune system, making cattle more prone to persistent colonization after re-infection. The RNA-Seq technology was used to quantify changes in the recto-anal junction (RAJ) and ileal Peyer’s patches (I+PP) transcriptome of nine Holstein-Friesian calves in response to a primary and secondary infection with EHEC O157:H7. A total of 21.046 genes were detected at least once in one of the nine RAJ or I+PP samples. We observed a downregulation of immunostimulatory genes and a stimulation of immunosuppressing genes. Summarized, the gene-expression in the I+PP is far more influenced by EHEC infection compared to the RAJ and a first EHEC infection modulates the immune response most likely promoting the colonization during a second infection.

Marta Maroszyńska is a PhD student, senior year, in the Institute of Fermentation Technology and Microbiology, Department of Food Science and Biotechnology, Lodz University of Technology, Poland. She is the scientific and research worker of Industrial Biotechnology Laboratory in BioNanoPark, Lodz Regional Park of Science and Technology. This center is the first in Poland witch unit their own laboratories in bio- and nanotechnologies and innovative start-ups

Candida species are both a normal member of microbiota of most mammals and an opportunistic pathogen. They are recognized as the most frequent etiological agent of systemic and invasive candidiasis in humans. In addition, the biofilm formed by the yeast is a serious problem in medicine and many industries. Therefore, searching for new antifungal substances that may be effective in preventing the spread of Candida yeast remains a big challenge. Our goal was to determine the antimicrobial activity of essential oils against clinical and food-born strains of Candida. Minimal inhibitory concentration (MIC) of five essential oils, cinnamon oil (Cinnamomum zeylanicum), tea tree oil (Melaleuca alternifolia), clove oil (Syzygium aromaticum), peppermint oil (Mentha piperita) and thyme oil (Thymus vulgaris) against seven clinical strains of Candida albicans and five food-borne strains: Candida lusitaniae, Candida famata, Candida krusei, Candida rugosa, Candida boidinii was determined by serial dilution method. All tested isolates were sensitive to all essential oils. MIC ranged from 0,031% to 4% for clinical isolates and from 0,031% to 12,5% for food-borne isolates. Peppermint oil showed the weakest activity while cinnamon oil demonstrated the strongest one. The activity rank of essential oils was as follows: cinnamon oil>thyme oil>clove oil>tea tree oil>peppermint oil. High activity of cinnamon oil can be explained by a high content, about 90%, of the main component cinnamaldehyde which is considered to be very active. Our study shows that the essential oils may be attractive antimicrobial agents with broad spectrum activity, chip and environmentally safe and potentially used to combat drug-resistant hospital superbugs

Carlos Quesada-Gómez is a lecturer on Anaerobic Bacteriology, School of Microbiology, University of Costa Rica. He has a M.Sc. in Medical Bacteriology and is currently a Ph.D. Candidate working with Clostridium difficile. His research group is working on the molecular characterization of different isolates by PFGE, toxinotyping, MLST and whole genome sequence methods which have made possible the description of the hipervirulent NAP1/027 and other novel genotypes in Costa Rica and Latin America. C. difficile pathogenesis is another area of study in which the level of virulence is being associated with the genotypes and its toxins using cellular and animal models.

Studies have shown that TcdB is necessary for Clostridium difficile associated infections. Variations in TcdB have been proposed to contribute to the virulence of epidemic NAP1/027 strains. Toxin B from NAP9/017 strains (A-B+) induces a variant cytopathic effect (CPE) on cells associated to differences in GTPases glucosylation. However, the impact of variations in substrate glucosylation by different C. difficile toxins B is unknown. The pathogenic potential associated to the glucosyltransferase activity of TcdB from NAP1, a NAP1 variant strain (NAP1V), VPI 10463 and NAP9 strains was determined. Ex vivo assays revealed that TcdB from NAP1 glucosylates RhoA, Rac1, Cdc42, Rap2A and R-Ras. TcdB from NAP1v and NAP9 do not glucosylate RhoA, whereas TcdB from VPI 10463 modifies only Rho GTPases. CPE evaluated over time in different cell lines showed that there were no differences in the percent of rounded cells induced by these toxins, while cell death assays revealed that TcdB that do not glucosylate RhoA induce a higher percent of cell death by necrosis. When TcdB was tested for its ability to induce TNF-α production by macrophages, NAP1 and VPI 10463 induced higher amounts of this cytokine at earlier times. Mouse ileal loops indicated that bacterial free supernatant and purified TcdB from NAP1 induced high levels of IL-1β. IL-6 levels were higher in loops inoculated with toxins that glucosylate RhoA. All together the results suggest that the broader spectrum of GTPases glucosylated by TcdB from NAP1 might be associated to the in vivo pathogenic potential. Furthermore, RhoA modification is not associated to cell death induce by variant toxins, but may be linked to production of TNF-α at earlier times and increase of IL-6 levels.

Sofia Nikolaki has a first degree in Physics, University of Crete and an MSC in Biotechnology from the Agricultural University of Athens. She has recently joint the MGM research group of the University of Patras, Greece as a PhD student and she is currently working on Microbial communities and pesticide interactions in oilseed and sunflower.

The Sahara region of Africa is the Earth’s largest source of aerosolizedsoil dust. It is estimated to contribute as much as one billion tons of soil per year to the global atmosphere. African dust is transported to the Mediterranean regionunder particular meteorological conditions occurring during spring and summer. These dust events, inject a huge amount of microorganisms like fungal spores, bacteria, archaea, and viruses. They affect visibility, climate, human health and the quality of life. Once these particles are in the atmosphere, transformation, transport and removal can take place. These processes depend on several factors, such as aerosol sizes, concentration and chemical composition, location and meteorological effects.Considering the importance of airborne microorganisms for human health, agricultural productivity and ecosystem stability, we assessed the bacterial composition during several sandstorm events in the South of Greece that occurred from January 2013 until the April 2014. In total twenty-three (23) samples has been characterized using Illumina amplicon sequencing. In more detail the fusion primers U341F and 805R were used to amplify theV3-V4 region of the 16S rRNA gene. Sequencing data will be analyzed using the bioinformatics tools QIIME and custom scripts. In more detaildata analysis involves the following: i) filtering of low quality sequences, ii) alignment of sequences (SILVA database), iii) detection and removal of chimeras, iv) clustering sequences into OTU with 97% sequence identity and v) taxonomic assignment of OTUs.Our results indicate that during the dust events the relative abundance of Alphaproteobacteria and more specifically members of the Rhizobiaceae family are rising. The results of the sequencing barcoded PCR amplicons will be presented in more detail and the correlations between airborne bacterial communities will be discussed.