Koronavirusi i 21. vek

Koronavirusi pripadaju RNK virusima, a naziv potiče od latinske reči “corona” usled slič- nosti sa vencem (koronom) sunčevih zraka. Sve do početka 21. veka, koronavirusi su povezi- vani sa sezonskim prehladama i nisu bili prepoznati kao značajni humani patogeni. Međutim, 2002. godine u Kini je identifikovan uzročnik teškog akutnog respiratornog sin- droma – SARS (engl. Severe acute respiratory syndrome), pri čemu su zabeleženi smrtni isho- di, a virus je označen kao SARS-CoV. Deset godina kasnije, 2012. godine, u Saudijskoj Arabiji identifikovan je MERS-CoV (engl. Middle East respiratory syndrome Coronavirus), a krajem 2019. godine i novi koronavirus koji je kasnije označen kao SARS-CoV-2 usled sličnosti sa SARS-CoV. Broj umrlih usled infekcije SARS-CoV-2 i razvoja bolesti COVID-19 (engl. coronavi- rus disease 2019) procenjuje se do sada na oko 6 miliona ljudi globalno. Usled brzog širenja SARS-CoV-2, Svetska Zdravstvena Organizacija objavila je 11. marta 2020. pandemiju, koja je još uvek na snazi. Pandemija je dovela do brzog odgovora na COVID-19 što je za posledicu imalo velika finansijska ulaganja u razvoj bolničkih kapaciteta, novih tera- pijskih opcija, a posebno na razvoj vakcina protiv COVID-19. Međutim, paralelno sa razvojem i primenom vakcina došlo je do mutacija u genomu SARS-CoV-2 i pojave novih varijanti virusa što je posledično dovelo do manje efikasnosti vakcina i dostupnih terapijskih opcija. U okviru izlaganja dat je uporedni pregled karakteristika virusa SARS-CoV, MERS-CoV i SARS-CoV-2. Pažnja koju su privukli koronavirusi 21. veka i iskustva i znanja stečena njiho- vom pojavom, omogući će bolji odgovor na potencijalno nove koronaviruse, kao i bolji odgo- vor na epidemije ili pandemije uzrokovane drugim patogenima

About the HPV vaccine: how it works and who can get it

Human papilloma viruses (HPV) are small non-enveloped DNA viruses that posses great affinity for epithelial tissue. So far, more than 200 genotypes of HPV have been identified, of which around 40 types are associated with genital infections in man and woman. HPV is the most sexually transmited virus in the world leading to the fact that HPV infection is the major cause of cervical cancer. Besides, HPV is thought to be responsible for more than 90% of anal cancer, 70% of vaginal and vulvar cancer, 70% of oropharingeal cancer and 60% of penile cancer [1, 2]. HPV vaccinationn is the most effective procedure in prevention of HPV-related cancers. Prophylactic 9-valent HPV vaccine which targets high-risk HPV types is the most preferable one and is available in many countries worlwide including Balkan region. The main goal of this presentation is to improve awareness of great importance of HPV vaccination in target popullations.5th Congress of Pharmacists of Bosnia and Herzegovina with international participation, Abstract book, „Competencies of pharmacists – from drug design to successful disease treatment“, Sarajevo, November 9-12, 2023.Predavanje po poziv

Isolation, cultivation, and in vitro susceptibility testing of Borrelia burgdorferi sensu lato: A review

Lyme borreliosis is the most common vector-borne disease in the northern hemisphere. The agents of Lyme borreliosis are borrelia, bacteria of the family Spirochaetaceae, which are grouped in Borrelia burgdorferi sensu lato species complex. Borreliae are fastidious, slow-growing and biochemically inactive bacteria that need special attention and optimal conditions for cultivation. The isolation of Borrelia from clinical material and their cultivation is a time-consuming and demanding procedure. Cultivation lasts from 9 up to 12 weeks, which is much longer than is necessary to grow most other human bacterial pathogens. Although B. burgdorferi sensu lato is susceptible to a wide range of antimicrobial agents in vitro, up to now the susceptibility of individual Borrelia species to antibiotics is defined only partially. [Projekat Ministarstva nauke Republike Srbije, br. 175011

Functional characterization of the CmbT transporter responsible for multidrug resistance on structurally different substrates of the strain Lactococcus lactis subsp. cremoris MG1363

Lactococcus lactis pripada grupi bakterija mlečne kiseline (BMK) i ima dugotrajnu primenu u prehrambenoj industriji kao starter kultura, najviše u proizvodnji tvrdih i polutvrdih sireva. Kako L. lactis ima ključnu ulogu u formiranju ukusa i teksture finalnog proizvoda, a doprinosi i produženju trajnosti namirnica, veliki ekonomski i industrijski značaj doveo je do toga da su laktokoke predmet stalnog izučavanja. Iako se generalno smatraju bezbednim mikroorganizmima, mnogi istraživači ukazuju na činjenicu da laktokoke, kao i druge bakterije mlečne kiseline mogu biti rezervoar gena za rezistenciju. U cilju sprečavanja širenja ovakvih gena na druge bakterije intestinalnog trakta ili patogene bakterije prisutne u hrani, od velikog značaja je izučavanje mehanizama rezistencije, posebno kod laktokoka namenjenih za prehrambenu industriju. Analizom genoma L. lactis detektovano je prisustvo 40 potencijalnih gena za MDR (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 novi MDR transporter u soju L. lactis. Gen cmbT identifikovan je po prvi put prilikom izučavanja regulacije metabolizma sumpora u L. lactis. Primenom različitih kompjuterskih programa, pokazano je da cmbT kodira transmembranski integralni efluks protein (veličine 454 aminokiseline) 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 i ekspresija cmbT gena pomoću precizno regulisanog NICE (eng. NIsin Controlled gene Expression) sistema...Lactococcus lactis is a lactic acid bacterium (LAB) widely used as a constituent of many industrial and artisanal starter cultures in dairy industry, especially for fermentation of hard and semi hard cheeses. This organism plays a key role in the formation of flavour and texture of cheese and its preservation and because of the great industrial importance it has been the subject of numerous studies. Although they have acquired the „Generaly Regarded As Safe“ (GRAS) status, many investigators have speculated that lactococci as well as other LAB may act as reservoirs of antibiotic resistance genes. The main threat associated with these bacteria is that they can transfer resistance genes to intestinal microorganisms and food-associated pathogenic bacteria. In order to prevent and reduce the spreading of the resistance genes, more studies of multidrug resistance (MDR) transporters in lactococci intended for use in food systems are needed. The genome analysis of L. lactis indicated the presence of at least 40 putative drug transporter genes, but only two ABC transporters, LmrA, and LmrCD, and one MFS transporter, LmrP, have been studied in detail and all three have demonstrated MDR activity experimentally. The aim of this thesis was to investigate and characterize new MDR transporter CmbT (Cysteine and Methionine Biosynthesis Transporter) in L. lactis. The cmbT gene was originally identified from a random mutagenesis study of cysteine and methionine biosynthesis regulation in L. lactis. This gene is predicted to encode an integral membrane efflux protein (454 amino acids) with homology to members of the 2.A.1.3.X drug/H+ antiporter-2 subfamily of the major facilitator superfamily (MFS). In order to follow the transport function of the CmbT protein, the cmbT gene was cloned and over-expressed in L. lactis NZ9000 using the nisin controlled gene expression (NICE) system..

Evaluation of novel compounds as anti-bacterial or anti-virulence agents

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

Carbapenem-Resistant Acinetobacter baumannii from Serbia: Revision of CarO Classification

Carbapenem-resistant A. baumannii present a significant therapeutic challenge for the treatment of nosocomial infections in many European countries. Although it is known that the gradient of A. baumannii prevalence increases from northern to southern Europe, this study provides the first data from Serbia. Twenty-eight carbapenem-resistant A. baumannii clinical isolates were collected at a Serbian pediatric hospital during a 2-year period. The majority of isolates (67.68%) belonged to the sequence type Group 1, European clonal complex II. All isolates harbored intrinsic OXA-51 and AmpC cephalosporinase. OXA-23 was detected in 16 isolates (57.14%), OXA-24 in 23 isolates (82.14%) and OXA-58 in 11 isolates (39.29%). Six of the isolates (21.43%) harbored all of the analyzed oxacillinases, except OXA-143 and OXA-235 that were not detected in this study. Production of oxacillinases was detected in different pulsotypes indicating the presence of horizontal gene transfer. NDM-1, VIM and IMP were not detected in analyzed clinical A. baumannii isolates. ISAba1 insertion sequence was present upstream of OXA-51 in one isolate, upstream of AmpC in 13 isolates and upstream of OXA-23 in 10 isolates. In silico analysis of carO sequences from analyzed A. baumannii isolates revealed the existence of two out of six highly polymorphic CarO variants. The phylogenetic analysis of CarO protein among Acinetobacter species revised the previous classification CarO variants into three groups based on strong bootstraps scores in the tree analysis. Group I comprises four variants (I-IV) while Groups II and III contain only one variant each. One half of the Serbian clinical isolates belong to Group I variant I, while the other half belongs to Group I variant III

LraI from Lactococcus raffinolactis BGTRK10-1, an Isoschizomer of EcoRI, Exhibits Ion Concentration-Dependent Specific Star Activity

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

Virulence traits associated with Burkholderia cenocepacia ST856 epidemic strain isolated from cystic fibrosis patients

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

Over-expressed CmbT multidrug resistance transporter improves the fitness of Lactococcus lactis

U ovom radu je izučavan uticaj povećane ekspresije cmbT gena, odgovornog za sintezu CmbT MDR transportera, na rast Lactococcus lactis. L. lactis pripada grupi bakterija mlečne kiseline (BMK) i ima veliku primenu u prehrambenoj industriji kao starter kultura. CmbT transporter je nedavno okarakterisan MDR protein soja L. lactis, koji doprinosi rezistenciji na različite toksične agense kao i na neke klinički značajne antibiotike. U ovom radu je cmbT gen višestruko eksprimiran u soju L. lactis NZ9000 dodavanjem nizina kao inducera. Povećana ekspresija cmbT gena je praćena metodom reverzne transkripcije (RT-PCR). Pokazano je da se nakon dodatka subinhibitornih koncentracija nizina u medijum za rast povećava količina sintetisane informacione RNK specifične za cmbT gen. Rast soja L. lactis NZ9000/pCT50, u kome je višestruko eksprimiran cmbT gen i L. lactis NZ9000 kontrolnog soja praćen je u bogatom i hemijski definisanom medijumu u prisustvu samo metionina (0.084 mM) ili kombinacije metionina i cisteina (8.4 mM i 8.2 mM). Praćene su krive rasta oba soja, a nakon izračunavanja odgovarajućih vremena generacije, rezultati su pokazali da L. lactis NZ9000/pCT50, brže raste u odnosu na kontrolni soj. Uočena razlika je najverovatnije posledica aktivnosti CmbT transportera koji doprinosi izbacivanju toksičnih agenasa iz ćelije i na taj način poboljšava adaptivne sposobnosti bakterije koja ga eksprimira i daje joj selektivnu prednost.The influence of the over-expression of CmbT multidrug resistance transporter on the growth rate of Lactococcus lactis NZ9000 was studied. L. lactis is a lactic acid bacteria (LAB) widely used as a starter culture in dairy industry. Recently characterized CmbT MDR transporter in L. lactis confers resistance to a wide variety of toxic compounds as well as to some clinically relevant antibiotics. In this study, the cmbT gene was over-expressed in the strain L. lactis NZ9000 in the presence of nisin inducer. Over-expression of the cmbT gene in L. lactis NZ9000 was followed by RT-PCR. The obtained results showed that the cmbT gene was successfully over-expressed by addition of sub-inhibitory amounts of nisin. Growth curves of L. lactis NZ9000/pCT50 over-expressing the cmbT gene and L. lactis NZ9000 control strain were followed in the rich medium as well as in the chemically defined medium in the presence solely of methionine (0.084 mM) or mix of methionine and cysteine (8.4 mM and 8.2 mM, respectively). Resulting doubling times revealed that L. lactis NZ9000/pCT50 had higher growth rate comparing to the control strain. This could be a consequence of the CmbT efflux activity, which improves the fitness of the host bacterium through the elimination of toxic compounds from the cell