Principal Investigator: Dr Branko Jovcic

            Among all of the bacterial drug resistance problems, Gram-negative pathogens are particularly worrisome, because they are becoming resistant to nearly all drugs that are being considered for the treatment. Causative agents of the most serious gram-negative infections (due to limited treatment options and a high mortality rates) are carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter. Globally, urgent actions are recognized as necessary in order to find novel antimicrobials that will diminish the threat of multidrug-resistant Gram-negative bacteria to global public health. So far, novel antibiotic producers were screened among soil Actinomycetales. However, we are now aware that novel ecological niches must be exploited and screening should be expended towards other bacterial species as potential antibiotic producers. Sediments of glacial lakes from Western Balkans are unexplored natural treasure and represent a challenge regarding their microbial genomic and metabolic potential. Remote high mountain lakes, being far from habitation and located in extreme environments, receive less impact from human activities but magnify the effects of global climate changes, and can thus be taken as a mirror of natural environmental changes. Therefore, exploiting their microbial is of crucial importance. During the project we will analyze sediments of three lakes in Montenegro and Bosnia and Herzegovina. Lakes will be selected due to possible anthropogenic influences - ranging from lakes with minimal anthropogenic impact to that with significant anthropogenic impact. In order to get the full insight into these microbial communities, both microbiological and metagenomic approach will be used for analyses. Cultivable bacteria will be isolated from sediments and grown in aerobic and anaerobic conditions on various media. Cultivated bacteria will be then subjected to testing of antimicrobial compunds production. 16S rDNAmetagenomic analysis will be performed in order to determine the microbial diversity in lake sediments. Functional metagenomic libraries will be constructed in order to fully exploit the genomic potential of microbial communities from sediments. Functional metagenomic libraries will be expressed in E. coli and tested for the production of compounds active against carbapenem-resistant, multidrug-resistant Gram-negatives in order to avoid selection of antimicrobials that are already known, and those that can not overcome existing resistance mechanisms that are globally dispersed among Gram-negative pathogens. Also, functional metagenomic libraries will be screened for the presence of various antibiotic-resistance genetic determinants. After the selection of cosmid that encompasses potentially novel antimicrobial compound it will be subjected to sequencing in order to reveal gene(s) involved in biosynthesis of this compound. Also, we will determine biochemical properties of a novel antimicrobial compound, perform its chemical purification, as well as analyze the interactions between the compound and potential target molecules. However, since sampling of glacial lake sediments is possible only in the summer period due to high altitudes of lake localities we will, during first six months of the project, analyze laboratory collection of Lactic Acid Bacteria (LAB) for the producers of antimicrobial peptides active against multidrug-resistant Gram-negative bacteria. This unique collection includes (LAB) isolated from fermented milk products produced in households of Western Balkan according to traditional recipes as well as strains of human origin. Screening will be performed by means of agar well diffusion assay, and compounds active against tested bacteria will be subjected to further biochemical characterization.