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Antimicrobial resistance is a global threat, leading to an alarming increase in the prevalence of bacterial infections that can no longer be treated with available antibiotics. The World Health Organization estimates that by 2050 up to 10 million deaths per year could be associated with antimicrobial resistance, which would equal the annual number of cancer deaths worldwide. To overcome this emerging crisis, novel anti-bacterial compounds are urgently needed. There are two possible approaches in the fight against bacterial infections: a) targeting structures within bacterial cells, similar to existing antibiotics; and/or b) targeting virulence factors rather than bacterial growth. Here, for the first time, we provide a comprehensive overview of the key steps in the evaluation of potential new anti-bacterial and/or anti-virulence compounds. The methods described in this review include: a) in silico methods for the evaluation of novel compounds; b) anti-bacterial assays (MIC, MBC, Time-kill); b) anti-virulence assays (anti-biofilm, anti-quorum sensing, anti-adhesion); and c) evaluation of safety aspects (cytotoxicity assay and Ames test). Overall, we provide a detailed description of the methods that are an essential tool for chemists, computational chemists, microbiologists, and toxicologists in the evaluation of potential novel antimicrobial compounds. These methods are cost-effective and have high predictive value. They are widely used in preclinical studies to identify new molecular candidates, for further investigation in animal and human trials.

Since the WHO declared the COVID-19 pandemic in March 2020, the disease has spread rapidly leading to overload of the health system and many of the patients infected with SARS-CoV-2 needed to be admitted to the intensive care unit (ICU). Around 10% of patients with the severe manifestation of COVID-19 need noninvasive or invasive mechanical ventilation, which represent a risk factor for Acinetobacter baumannii superinfection. The 64 A. baumannii isolates were recovered from COVID-19 patients admitted to ICU at General Hospital “Dr Laza K. Lazarević” Šabac, Serbia, during the period from December 2020 to February 2021. All patients required mechanical ventilation and mortality rate was 100%. The goal of this study was to evaluate antibiotic resistance profiles and virulence potential of A. baumannii isolates recovered from patients with severe form of COVID-19 who had a need for mechanical ventilation. All tested A. baumannii isolates (n = 64) were sensitive to colistin, while resistant to meropenem, imipenem, gentamicin, tobramycin, and levofloxacin according to the broth microdilution method and MDR phenotype was confirmed. In all tested isolates, representatives of international clone 2 (IC2) classified by multiplex PCR for clonal lineage identification, blaAmpC, blaOXA-51, and blaOXA-23 genes were present, as well as ISAba1 insertion sequence upstream of blaOXA-23. Clonal distribution of one dominant strain was found, but individual strains showed phenotypic differences in the level of antibiotic resistance, biofilm formation, and binding to mucin and motility. According to PFGE, four isolates were sequenced and antibiotic resistance genes as well as virulence factors genes were analyzed in these genomes. The results of this study represent the first report on virulence potential of MDR A. baumannii from hospital in Serbia.

Colistin is used as a “last resort” drug in the treatmentof carbapenem-resistant Acinetobacter baumanniiinfections. Though not the most convenient option due tothe serious side effects, this antibiotic can be very efficientif administered properly (Garnacho-Montero and Timsit,2019). However, for several years now, we are witnessinga steady emergence of colistin-resistant (ColR) strains, withthe constant threat of a sudden burst worldwide (Nowak etal., 2017). Having in mind that these strains are usuallynon-susceptible to any of the existing treatments, i.e.pandrug-resistant (PDR), the development of newtherapeutic strategies is desperately needed.In the present study, we showed that colistinsusceptibility of ColR A. baumannii strains can be fullyrestored in the presence of very low amounts of seleniumnanoparticles (SeNPs). To demonstrate potent synergisticinteractions between colistin and SeNPs, we performedcheckerboard and time-kill analyses.

Trimethoprim-sulfamethoxazole (SXT) is the preferable treatment option of the infections caused by Achromobacter spp. Our study aimed to analyze the SXT resistance of 98 Achromobacter spp. isolates from pediatric patients, among which 33 isolates were SXT-resistant. The presence of intI1 was screened by PCR and genome sequence analyses. The intI1 gene was detected in 10 of SXT-resistant isolates that had shorter intI1 PCR fragments named intI1S. Structural changes in intI1S were confirmed by genome sequencing and analyses which revealed 86 amino acids deletion in IntI1S protein compared to canonical IntI1 protein. All IntI1S isolates were of non-CF origin. Pan-genome analysis of intI1S bearing A. xylosoxidans isolates comprised 9052 genes, with the core genome consisting of 5455 protein-coding genes. Results in this study indicate that IntI1S isolates were derived from clinical settings and that cystic fibrosis (CF) patients were potential reservoirs for healthcare-associated infections that occurred in non-CF patients.

Aims To investigate the synergistic activity of colistin and selenium nanoparticles (SeNPs) against pandrug-resistant (PDR) Ac. baumannii. Methods and Results Chequerboard and time-kill assays were employed to explore the potential synergistic interactions between colistin and SeNPs against Ac. baumannii isolates (8), previously determined as colistin-resistant (MIC range 16-256 mu g ml(-1)). Also, whole-genome sequencing (WGS) and gene expression analyses were used to elucidate the mechanisms of colistin resistance. Exceptionally strong synergistic activity (FICI range 0.004-0.035) of colistin and SeNPs against colistin-resistant isolates was revealed. Colistin (0.5 or 1 mu g ml(-1)) used in combination with SeNPs (0.5 mu g ml(-1)) was able to reduce initial inoculum during the first 4 h of incubation, in contrast to colistin (0.5, 1 or 2 mu g ml(-1)) alone. Conclusions These findings propose colistin/SeNPs combination as a new option to fight PDR Ac. baumannii, the therapeutic possibilities of which should be proved in future in vivo studies. Significance and Impact of Study Here we present the first evidence of synergy between colistin and selenium compounds against bacteria in general. Also, WGS and gene expression analyses provide some new insights into Ac. baumannii colistin resistance mechanisms.

Vaccination is one of the most efficient strategies for prevention of infection diseases, but with introduction of sub-unit vaccines with lower immunogenicity adjuvants were needed to enhance the immune response. The term adjuvant is from Latin verb adjuvare which means „to aid”. Adjuvants have been used in vaccines for more than 90 years. The longest adjuvant history belongs to aluminium salts, but novel adjuvants have been introduced in licensed vaccines in last 30 years. These novel adjuvants are AS04, which consists of aluminium hydroxide and Toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A and is used in hepatitis B vaccine Fendrix® and HPV vaccine Cervarix®, emulsion based adjuvants which are part of several influenza vaccines-MF59 (Fluad® and Focetria®) and AS03 (Pandemrix®), AS01 liposomal adjuvant which is combination of two distinct immunostimulatory molecules and is component of herpes zoster and malaria vaccine and virosomes included in hepatits A vaccine (Epaxal®) and influenza vaccines (Inflexal® V and Invivac®). Adjuvant development and better insight into their mechanism of action are of great importance in order to replace empirical with rational use of adjuvants, without affecting vaccine safety., Vakcinacija je jedna od najznačajnijih strategija za prevenciju infektivnih oboljenja, a razvoj subjediničnih vakcina doveo je do potrebe za primenom adjuvanasa u vakcinama. Naziv adjuvans potiče od latinske reči adjuvare što znači "pomoći". Adjuvansi su supstance koje se primenjuju u vakcinama više od 90 godina, a dodaju se da bi se povećala imunogenost antigena koji imaju nizak imunostimulatorni potencijal. U vakcinama za primenu kod ljudi, najduže se kao adjuvansi koriste soli aluminijuma, ali je poslednjih decenija nekoliko novih adjuvanasa uključeno u vakcine koje su odobrene za primenu. Adjuvantni sistem AS04, ulazi u sastav vakcina protiv humanog papiloma virusa (Cervarix®) i hepatitis B virusa (Fendrix®) i sadrži aluminijum hidroksid i TLR4 agonist, monofosforil lipid A. Zatim, adjuvansi na bazi emulzije (MF59 i AS03) su sastavni deo vakcina protiv gripa (Fluad®, Focetria® i Pandemrix®). Kombinacija dva imunostimulatorna molekula, označena kao AS01, ulazi u sastav vakcine protiv herpes zoster virusa i malarije dok se adjuvansi na bazi virozoma koriste u vakcinama protiv hepatitisa A (Epaxal®) i gripa (Inflexal® V, Invivac®). Daljim razvojem adjuvanasa, i ispitivanjem njihovog mehanizma delovanja oni će se umesto empirijski, sve više koristili racionalno i ciljano, čime će se postići bolji imunogeni profil vakcina, bez narušavanja njihovog bezbednosnog profila.

Cancer is one of the leading causes of morbidity and mortality worldwide and great efforts are underway to develop new therapeutic protocols. One of the approaches is immunotherapy which uses the immune system and its components to fight against cancer. The two main axes of cancer immunotherapy refer to passive and active treatments. Passive immunotherapy includes administration of tumor-specific antibodies and autologous T cells which destroy tumor cells, while active immunotherapy is directed at inducing the patient´s own antitumor immune responses and refers to cancer vaccines and immune checkpoint inhibitors. Vaccination of tumor-bearing individuals with tumor cells/antigens or autologous dendritic cells pulsed with tumor antigens may result in enhanced antitumor immune response. However, vaccine design is a complex, multi-component task, and the optimal combinations of antigens, adjuvants, delivery methods and routes of administration need to be precisely defined. Active immunotherapy also addresses the immunosuppressive and tolerogenic mechanisms developed by tumors. This review provides an overview of new results from clinical studies of therapeutic cancer vaccines and discusses their implications for the clinical use, alone or in combination with other immunotherapeutic strategies., Maligni tumor (rak, karcinom) je jedan od vodećih uzroka obolevanja i smrtnosti pa se veliki napori ulažu u razvoj novih terapijskih pristupa. Savremena imunoterapija malignih tumora obuhvata primenu antitumorskih antitela i autologih T ćelija koje uništavaju ćelije tumora (pasivna imunoterapija) i pojačanje slabog antitumorskog imunskog odgovora domaćina (aktivna imunoterapija) vakcinacijom i primenom antitela koja blokiraju inhibitorne receptore (kontrolne tačke) T limfocita. Vakcinacija pacijenata obolelih od tumora njihovim sopstvenim tumorskim ćelijama, antigenima tih ćelija ili dendritskim ćelijama koje su inkubirane sa tumorskim antigenima stimuliše imunski sistem pacijenta da prepozna tumorske antigene i eliminiše maligne ćelije. Međutim, razvoj terapijskih tumorskih vakcina suočen je sa brojnim izazovima vezanim za njihov dizajn, u smislu optimalne kombinacije antigena, adjuvansa i nosača, kao i za način primene. Pored toga, savremena aktivna imunoterapija treba da prevaziđe nisku imunogenost tumora i imunosupresivne mehanizme mikrosredine tumora kod pacijenata sa klinički ispoljenom bolesti. U ovom radu prikazani su rezultati novijih kliničkih studija u kojima su ispitivane različite terapijske vakcine za karcinom i diskutovano je o njihovoj mogućoj primeni u kliničkoj praksi, kako samih, tako i u kombinaciji sa drugim imunoterapijama.

Screening for producers of potent antimicrobial peptides, resulted in the isolation of Bacillus cereus BGNM1 with strong antimicrobial activity against Listeria monocytogenes. Genome sequence analysis revealed that BGNM1 contains the gene cluster associated with the production of the lantibiotic, thusin, previously identified in B. thuringiensis. Purification of the antimicrobial activity confirmed that strain BGMN1 produces thusin. Both thusin sensitive and resistant strains were detected among clinical isolates of Streptococcus agalactiae. Random mutagenesis of a thusin sensitive strain, S. agalactiae B782, was performed in an attempt to identify the receptor protein for thusin. Three independent thusin resistant mutants were selected and their complete genomes sequenced. Comparative sequence analysis of these mutants with the WT strain revealed that duplication of a region encoding a 79 amino acids repeat in a C-protein α-antigen was a common difference, suggesting it to be responsible for increased resistance to thusin. Since induced thusin resistant mutants showed higher level of resistance than the naturally resistant B761 strain, complete genome sequencing of strain B761 was performed to check the integrity of the C-protein α-antigen-encoding gene. This analysis revealed that this gene is deleted in B761, providing further evidence that this protein promotes interaction of the thusin with receptor.

Plasmids are autonomous episomally replicating genetic elements, which carry backbone genes important for the replication and maintenance within their host, and accessory genes that might confer an advantage to their host under specific selective pressure in its ecological niche. The genome of dairy isolate L. lactis subsp. lactis bv. diacetylactis S50 was sequenced using the PacBio SMRT Cell Seq-RSII platform and revealed to possess one of the largest plasmidomes among L. lactis strains studied so far, harboring six plasmids: pS6 (5553 bp), pS7a (7308 bp), pS7b (7266 bp), pS19 (19,027 bp), pS74 (74,256 bp) and pS127 (127,002 bp) in total representing 8.9% of genome size (240,412 bp). Based on predicted plasmid replication proteins and origins it appears that all six plasmids replicate via the theta-type mechanism. The two the largest plasmids (pS74 and pS127), carry a number of genes known to be important for growth and survival in the dairy environment. These genes encode technological functions such as bacteriocin production, protein degradation, magnesium and cobalt/nickel transporters, selenium binding, exopolysaccharides (EPS) production, bacteriophage and stress resistance. Beside genes for replication, the small plasmids (pS6, pS7a, pS7a, and pS19) also carry genes important for mobilization and host survival such as type I restriction-modification (R-M) system, metal transporters, enzymes and transcriptional regulators. All plasmids in S50 strain are mobilizable, containing an oriT sequences, while pS127 is self-conjugative and allows for mobilization of the other plasmids. Small plasmids are prone to structural and segregational instability, while pS127 appeared to be segregationally stable thanks to the possession of two partition systems. The main characteristic of plasmid pS74 is EPS production, while plasmid pS127 is characterized by proteinase and multiple bacteriocins, tra locus, phage abortive systems and metal transporters. In addition to LcnA and

Screening for producers of potent antimicrobial peptides, resulted in the isolation of Bacillus cereus BGNM1 with strong antimicrobial activity against Listeria monocytogenes. Genome sequence analysis revealed that BGNM1 contains the gene cluster associated with the production of the lantibiotic, thusin, previously identified in B. thuringiensis. Purification of the antimicrobial activity confirmed that strain BGMN1 produces thusin. Both thusin sensitive and resistant strains were detected among clinical isolates of Streptococcus agalactiae. Random mutagenesis of a thusin sensitive strain, S. agalactiae B782, was performed in an attempt to identify the receptor protein for thusin. Three independent thusin resistant mutants were selected and their complete genomes sequenced. Comparative sequence analysis of these mutants with the WT strain revealed that duplication of a region encoding a 79 amino acids repeat in a C-protein alpha-antigen was a common difference, suggesting it to be responsible for increased resistance to thusin. Since induced thusin resistant mutants showed higher level of resistance than the naturally resistant B761 strain, complete genome sequencing of strain B761 was performed to check the integrity of the C-protein alpha-antigen-encoding gene. This analysis revealed that this gene is deleted in B761, providing further evidence that this protein promotes interaction of the thusin with receptor.

The antimicrobial susceptibility testing was conducted on 174 single isolates from poultry farms in Serbia and it was determined that seven Salmonella spp. were multidrug resistant. Sixteen serotypes were detected, but only serotype Infantis confirmed reduced susceptibility to colistin. Seven colistin resistant Salmonella Infantis were studied in detail using the WGS approach. Three sequence types were identified corresponding to different epizootiology region. The isolate from the Province of Vojvodina 3842 and isolates from Jagodina (92 and 821) are represented by the sequence type ST413 and ST11, respectively. Four isolates from Kraljevo are ST32, a common S. Infantis sequence type in humans, poultry and food. The fosfomycin resistance gene fosA7 in isolate 3842 and the vgaA gene in isolate 8418/2948 encoding resistance to pleuromutilins were reported for the first time in serovar Infantis. The changes in relative expression of the phoP/Q, mgrB and pmrA/B genes were detected. Single nucleotide polymorphisms of the pmrB gene, including transitions Val164Gly or Val164Met, and Arg92Pro are described. Analyses of quinolone resistance determining region revealed substitutions Ser83Tyr in GyrA protein and Thr57Ser and Ser80Arg in ParC protein. Based on WGS data, there are two major clusters among analyzed Salmonella Infantis isolates from central Serbia.

Long-term overuse of antibiotics has driven the propagation and spreading of antibiotic resistance genes (ARGs) such as efflux pumps in the environment, which can be transferred to clinically relevant pathogens. This study explored the abundance and diversity of ARGs and mobile genetic elements within bacterial communities from sediments of three Western Balkans glacial lakes: Plav Lake (high impact of human population), Black Lake (medium impact of human population) and Donje Bare Lake (remote lake, minimal impact of human population) via shotgun metagenomics. Assembled metagenomic sequences revealed that Resistance-Nodulation-Division (RND) efflux pumps genes were most abundant in metagenome from the Plav Lake. The Integron Finder bioinformatics tool detected 38clusters ofattCsiteslackingintegron-integrases (CALIN) elements: 20 from Plav Lake, four from Black Lake and 14 from Donje Bare Lake. A complete integron sequence was recovered only from the assembled metagenome from Plav Lake. Plasmid contents within the metagenomes were similar, with proportions of contigs being plasmid-related: 1.73% for Plav Lake, 1.59% for Black Lake and 1.64% for Donje Bare Lake. The investigation showed that RNDs and mobile genetic elements content correlated with human population impact.

Achromobacter spp. are recognized as emerging pathogens in hospitalized as well as in cystic fibrosis (CF) patients. From 2012 to 2015, we collected 69 clinical isolates (41 patient) of Achromobacter spp. from 13 patients with CF (CF isolates, n = 32) and 28 patients receiving care for other health conditions (non-CF isolates, n = 37). Molecular epidemiology and virulence potential of isolates were examined. Antimicrobial susceptibility, motility, ability to form biofilms and binding affinity to mucin, collagen, and fibronectin were tested to assess their virulence traits. The nrdA gene sequencing showed that A. xylosoxidans was the most prevalent species in both CF and non-CF patients. CF patients were also colonized with A. dolens/A, ruhlandii, A. insuavis, and A. spiritinus strains while non-CF group was somewhat less heterogenous, although A. insuavis, A. insolitus, and A. piechaudii strains were detected beside A. xylosoxidans. Three strains displayed clonal distribution, one among patients from the CF group and two among non-CF patients. No significant differences in susceptibility to antimicrobials were observed between CF and non-CF patients. About one third of the isolates were classified as strong biofilm producers, and the proportion of CF and non-CF isolates with the ability to form biofilm was almost identical. CF isolates were less motile compared to the non-CF group and no correlation was found between swimming phenotype and biofilm formation. On the other hand, CF isolates exhibited higher affinity to bind mucin, collagen, and fibronectin. In generall, CF isolates from our study exhibited in vitro properties that could be of importance for the colonization of CF patients.

16S rRNA gene-based metagenomic approach was used to assess the biodiversity of bacterial communities in the sediments of selected glacial lakes in the Western Balkans and to assess the impact of human population on these microbial communities. Sediment samples were collected from three glacial lakes, viz., Plav Lake (in a zone of the highest impact of human population), Black Lake (a zone of medium impact of human population), and Donje Bare Lake (a remote lake with minimal impact of human population). Canonical correlation analysis analysis indicated correlation between the distance of the lake from urbanized population and bacterial diversity in Donje Bare Lake sediment. Bacterial diversity of Black Lake sediment was correlated with high content of phosphorous and pH value. Chemical compounds exhibiting the most prominent correlation with bacterial diversity of Plav Lake were NH4-N, K2O, CaCo3, and total nitrogen . Additionally, CCA analysis indicated that population density was correlated with biodiversity of bacterial communities in Plav Lake sediment, which is the most exposed to human population. Multivariate regression revealed the highest correlation between the presence of Proteobacteria classes and population density and levels of NH4-N. The influence of human population was observed to be important for shaping the sediment communities in addition to biological and chemical factors.

Pseudomonas aeruginosa, which is a clinically important representative of Pseudomonas spp., has been recognized as causative agent of severe nosocomial infections worldwide. An increase in antibiotic resistance of P. aeruginosa clinical strains could be attributed to their capacity to acquire resistance through mobile genetic elements such as mobile integrons that are present in one-half of multidrug-resistant P. aeruginosa strains. Mobile class 1 integrons are recognized as genetic elements involved in the rapid dissemination of multiple genes encoding for antibiotic resistance. The LexA protein is a major repressor of integrase transcription, but differences in transcription regulation among bacterial species have also been noted. In this study, the promoter activity of class 1 integron integrase gene (intI1) and its variant lacking the LexA binding site in Pseudomonas putida WCS358 wild type, rpoS and psrA was analysed. The results show that the activity of the intI1 gene promoter decreased in the rpoS and psrA mutants in the stationary phase of growth compared to the wild type, which indicates the role of RpoS and PsrA proteins in the positive regulation of integrase transcription. Additionally, it was determined that the activity of the lexA gene promoter decreased in rpoS and psrA, and thus, we propose that PsrA indirectly regulates the intI1 gene promoter activity through regulation of lexA gene expression in co-operation with some additional regulators. In this study, intI1 gene expression was shown to be controlled by two major stress response (SOS and RpoS) regulons, which indicates that integrase has evolved to use both systems to sense the cell status.

Bacteria active against multi-drug resistant pathogens, isolated by direct selection of colonies from clover silage samples, produce zones of inhibition against two Gram-negative (Klebsiella pneumoniae Ni9 and Pseudomonas aeruginosa MMA83) and two Gram-positive (Staphylococcus aureus ATCC25923 and Listeria monocytogenes ATCC19111) pathogens. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 produced the largest zones of inhibition against all four pathogens when grown in LB broth with aeration at 37 degrees C. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 were identified as Brevibacillus laterosporus and pulsed field gel electrophoresis and extracellular protein profiles showed that three different strains (BGSP7, BGSP9 and BGSP11) were isolated. A semi-native SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) gel overlay assay showed that BGSP7 and BGSP9 produce small antimicrobial molecules of about 1.5 kDa, while BGSP11 produces antimicrobial molecules of 1.5 and 6 kDa active against S. aureus ATCC25923. Amino acid analysis of two antimicrobial molecules (1583.73 Da; from BGSP7 and 1556.31 Da; from BGSP11) revealed that they have a similar composition and differ only by virtue of the presence of a methionine which is present only in BGSP11 molecule. Genome sequencing of the three isolates revealed the presence of gene clusters associated with the production of non-ribosomally synthesized peptides (brevibacillin, bogorol, gramicidin S, plipastatin and tyrocin) and bacteriocins (laterosporulin, a lactococcin 972-like bacteriocin, as well as putative linocin M18, sactipeptide, UviB and lantipeptide-like molecules). Ultimately, the purification of a number of antimicrobial molecules from each isolate suggests that they can be considered as potent biocontrol strains that produce an arsenal of antimicrobial molecules active against Gram-positive and Gram-negative multi-resistant pathogens, fungi and insects.

Restriction enzymes are the main defence system against foreign DNA, in charge of preserving genome integrity. Lactococcus raffinolactis BGTRK10-1 expresses LraI Type II restriction-modification enzyme, whose activity is similar to that shown for EcoRI; LraI methyltransferase protects DNA from EcoRI cleavage. The gene encoding LraI endonuclease was cloned and overexpressed in E. coli. Purified enzyme showed the highest specific activity at lower temperatures (between 13 degrees C and 37 degrees C) and was stable after storage at -20 degrees C in 50% glycerol. The concentration of monovalent ions in the reaction buffer required for optimal activity of LraI restriction enzyme was 100 mM or higher. The recognition and cleavage sequence for LraI restriction enzyme was determined as 5'-G/AATTC-3', indicating that LraI restriction enzyme is an isoschizomer of EcoRI. In the reaction buffer with a lower salt concentration, LraI exhibits star activity and specifically recognizes and cuts another alternative sequence 5'-A/AATTC-3', leaving the same sticky ends on fragments as EcoRI, which makes them clonable into a linearized vector. Phylogenetic analysis based on sequence alignment pointed out the common origin of LraI restriction-modification system with previously described EcoRI-like restriction-modification systems.

Lactic acid bacteria (LAB) are widely used in the food industry, especially in the pro­duction of fermented dairy products and meat. The most studied species among LAB is Lactococcus lactis. L. lactis strains are of great importance in the production of fermented dairy products such as yogurt, butter, fresh cheese and some kind of semi-hard cheese. Although L. lactis acquired the 'Generally Regarded As Safe' (GRAS) status, many investigations indicated that lactococci may act as reservoirs of antibiotic resistance genes, which could be transferred to other bacterial species in human gastrointestinal tract includ­ing pathogens. The genome analysis of L. lactis indicated the presence of at least 40 putative drug transporter genes, and only four multidrug resistance (MDR) transporters are functional­ly characterized: LmrA, LmrP, LmrCD i CmbT. LmrA is the first described MDR transport­er in prokaryotes. LmrCD is responsible for resistance to cholate, which is an integral part of human bile and LmrCD is important for intestinal survival of lactococci that are used as probiotics. Secondary multidrug transporter LmrP confers resistance to lincosamides, mac­rolides, streptogramins and tetracyclines. CmbT protein has an effect on the host cell re­sistance to lincomycin, sulfadiazine, streptomycin, rifampicin, puromycin and sulfametox­azole. Since the food chain is an important way of transmitting resistance genes in human and animal population, it is of great importance to study the mechanisms of resistance in lactococci and other LAB, intended for the food industry., Bakterije mlečne kiseline (BMK) imaju veliku primenu u prehrambenoj industriji, posebno u proizvodnji fermentisanih mlečnih proizvoda i mesa. Najviše izučavana vrsta u okviru BMK je Lactococcus lactis. Sojevi koji pripadaju vrsti L. lactis su od izuzetnog značaja u proizvodnji fermentisanih mlečnih proizvoda poput kiselog mleka, maslaca, svežih sireva i nekih vrsta polutvrdih sireva. Iako se generalno laktokoke smatraju bezbednim mikroorganizmima, istraživanja ukazuju da mogu biti rezervoari gena za rezistenciju na antibiotike, koji se horizontalnim transferom mogu preneti na druge bakterijske vrste, uključujući i patogene. Pokazano je da se u membrani bakterija vrste L. lactis nalaze efluksne pumpe koje obezbeđuju rezistenciju na strukturno različite supstrate (eng. multidrug resistance transporters-MDR). Analizom genoma L. lactis detektovano je prisustvo 40 potencijalnih gena koji kodiraju MDR transportere, od čega su do danas svega četiri funkcionalno okarakterisana: LmrA, LmrP, LmrCD i CmbT. LmrA je prvi uopšte opisan primarni transporter kod prokariota. LmrCD obezbeđuje rezistenciju na holat koji ulazi u sastav žuči, što je značajno za preživljavanje laktokoka koje se primenjuju kao probiotici u digestivnom traktu ljudi i životinja. LmrP je sekundarni transporter i obezbeđuje rezistenciju na neke linkozamide, makrolide, streptogramine I tetracikline. CmbT je poslednji okarakterisan MDR transporter kod L. lactis i doprinosi rezistenciji na linkomicin, sulfonamidne antibiotike, sulfametoksazol i sulfadiazin, aminoglikozidni antibiotik streptomicin, aminonukleozidni antibiotik puromicin i semisintetski antibiotik rifampicin. Kako lanac hrane predstavlja važan put prenosa gena za rezistenciju u okviru ljudske i životinjske populacije, neophodno je intenzivno izučavanje mehanizama rezistencije kod laktokoka i drugih BMK namenjenih za upotrebu u prehrambenoj industriji.

Background: Burkholderia cenocepacia is considered one of the most problematic cystic fibrosis (CF) pathogens. Colonization prevalence in the Serbian CF population is high and virtually exclusively limited to a single highly transmissible clone of B. cenocepacia ST856 which is positive for both the B. cepacia epidemic strain marker (BCESM) and cable pilin, and is closely related to the epidemic strain CZ1 (ST32). Methods: Biofilm formation for 182 isolates, and adhesion to components of the host extracellular matrix, proteolytic activity, mucoidy and motility of selected ST856 representatives, as well as B. cenocepacia ST858 and ST859, and B. stabilis ST857, novel STs isolated from Serbian CF patients, were investigated in this study. The presence of the cepI, cepR, fliG, llpE, wbiI, and bcscV genes was analyzed. Results: Biofilm-formation ability of analyzed strains was poor under standard laboratory conditions, but changed in stress conditions (cold stress) and conditions that mimic CF milieu (increased CO2). All strains expressed ability to bind to collagen and fibronectin albeit with different intensity. Representatives of ST856 exhibited gelatinase activity. ST858, ST859 and 9/11 of ST856 genotypes were positive for swimming and twitching motility whereas ST857 was non-motile. Mucoidy was demonstrated in all ST856 genotypes, ST857 was semi-mucoid, and ST858 and ST859 were non-mucoid. Molecular analysis for major virulence factors revealed that ST856 and ST857 carried the six analyzed genes, while ST858 and ST859 were negative for the llpE gene. Conclusion: Variations in virulence phenotypes in different genotypes of epidemic B. cenocepacia ST856 clone, in vitro, could be a consequence of diversification driven by pathoadaptation. Diversity of epidemic clone genotypes virulence, could be challenging for accurate diagnosis and treatment, as well as for infection control.

Lactococcus lactis pripada grupi bakterija mlečne kiseline (BMK) i imadugotrajnu primenu u prehrambenoj industriji kao starter kultura, najviše u proizvodnjitvrdih i polutvrdih sireva. Kako L. lactis ima ključnu ulogu u formiranju ukusa iteksture finalnog proizvoda, a doprinosi i produženju trajnosti namirnica, velikiekonomski i industrijski značaj doveo je do toga da su laktokoke predmet stalnogizučavanja. Iako se generalno smatraju bezbednim mikroorganizmima, mnogiistraživači ukazuju na činjenicu da laktokoke, kao i druge bakterije mlečne kiselinemogu biti rezervoar gena za rezistenciju. U cilju sprečavanja širenja ovakvih gena nadruge bakterije intestinalnog trakta ili patogene bakterije prisutne u hrani, od velikogznačaja je izučavanje mehanizama rezistencije, posebno kod laktokoka namenjenih zaprehrambenu industriju.Analizom genoma L. lactis detektovano je prisustvo 40 potencijalnih gena zaMDR (eng. multidrug resistance) transportere, od čega su svega dva ABC transportera,LmrA i LmrCD i jedan MFS transporter, LmrP funkcionalno okarakterisani.Istraživanja prikazana u ovom radu su imala za cilj da se okarakteriše CmbT(eng. Cysteine and Methionine Biosynthesis Transporter) protein, potencijalno noviMDR transporter u soju L. lactis.Gen cmbT identifikovan je po prvi put prilikom izučavanja regulacijemetabolizma sumpora u L. lactis. Primenom različitih kompjuterskih programa,pokazano je da cmbT kodira transmembranski integralni efluks protein (veličine 454aminokiseline) koji pokazuje homologiju sa članovima 2.A.1.3.X supstrat/H+ antiporter-2 subfamilije.Prvi korak u funkcionalnoj karakterizaciji CmbT proteina bio je kloniranje iekspresija cmbT gena pomoću precizno regulisanog NICE (eng. NIsin Controlled gene Expression) sistema..., Lactococcus lactis is a lactic acid bacterium (LAB) widely used as a constituentof many industrial and artisanal starter cultures in dairy industry, especially forfermentation of hard and semi hard cheeses. This organism plays a key role in theformation of flavour and texture of cheese and its preservation and because of the greatindustrial importance it has been the subject of numerous studies. Although they haveacquired the „Generaly Regarded As Safe“ (GRAS) status, many investigators havespeculated that lactococci as well as other LAB may act as reservoirs of antibioticresistance genes. The main threat associated with these bacteria is that they can transferresistance genes to intestinal microorganisms and food-associated pathogenic bacteria.In order to prevent and reduce the spreading of the resistance genes, more studies ofmultidrug resistance (MDR) transporters in lactococci intended for use in food systemsare needed.The genome analysis of L. lactis indicated the presence of at least 40 putativedrug transporter genes, but only two ABC transporters, LmrA, and LmrCD, and oneMFS transporter, LmrP, have been studied in detail and all three have demonstratedMDR activity experimentally.The aim of this thesis was to investigate and characterize new MDR transporterCmbT (Cysteine and Methionine Biosynthesis Transporter) in L. lactis.The cmbT gene was originally identified from a random mutagenesis study ofcysteine and methionine biosynthesis regulation in L. lactis. This gene is predicted toencode an integral membrane efflux protein (454 amino acids) with homology tomembers of the 2.A.1.3.X drug/H+ antiporter-2 subfamily of the major facilitatorsuperfamily (MFS).In order to follow the transport function of the CmbT protein, the cmbT genewas cloned and over-expressed in L. lactis NZ9000 using the nisin controlled gene expression (NICE) system...