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The increasing prevalence of multidrug-resistant (MDR) Acinetobacter baumannii limits effective therapeutic options, and tigecycline has been considered one of the last resort therapies for MDR A. baumannii infections. Nevertheless, A. baumannii isolates resistant to tigecycline are becoming increasingly reported, mostly due to overexpression of efflux pumps. The three major RND efflux systems conferring tigecycline resistance in A. baumannii are AdeABC, AdeFGH, and AdeIJK, and their expression is regulated by the two-component system AdeRS, the LysR-type regulator AdeL, and the TetR-type regulator AdeN, respectively. Following the above, we aimed to determine the role of efflux pumps in tigecycline resistance of thirty-seven A. baumannii isolates collected from Western Balkan healthcare settings (Serbia, Bosnia and Herzegovina and Montenegro) in 2016 and 2022. The majority of isolates belonged to the most prevalent international clonal lineage IC2 (n = 32), four isolates are members of IC1, while only one isolate is identified as IC3. All tested isolates demonstrated a significant decrease in tigecycline MIC in presence of efflux pump inhibitor CCCP (≥16-fold reduction) indicating that mechanism responsible for tigecycline resistance is antibiotic efflux. The comparison of target efflux pump regulatory proteins, translated from nucleotide sequences, to reference strains ATCC19606 and ATCC17978 revealed that most of the isolates have G186V and N268H alternations in AdeS (n = 32), while most common changes in AdeR were V120I and A136V (n = 29) as described in previous studies. Substitution Q262R was detected exclusively in AdeL proteins of IC1 isolates, while no mutations were observed within AdeN regulators. Expression of the adeB, adeG, and adeJ genes in six selected isolates was upregulated in four (1,4- to 3-fold), six (1,6- to 2,6-fold), and three isolates (1,7- to 4-fold), respectively. This study confirmed that overexpression of efflux pump encoding genes enab

During the past decades several quorum sensing inhibitors (QSI) of plant origin have been isolated and chemically characterized. QSI agents of plant origin represent potential alternative or complementary approach to antibiotic treatment of multidrug-resistant bacteria and infections caused by bacterial biofilms. The aim of the current study was to screen QSI activities of horseradish root extracts obtained using different organic solvents and different root processing methods (drying at 40°C, 60°C or extraction of fresh material). Common opportunistic pathogen Pseudomonas aeruginosa MMA83 was used for QSI screen. RT-qPCR was used to analyze the effect of the extract on the relative mRNA levels of the genes QS (lasR, lasI, rhlR, rhlI, mvfR, pqsH) and the genes involved in P. aeruginosa MMA83 virulence (lasB, phzM, rhlC, algK, pvdS). Chemical composition of extracts was determined by UHPLC Q-ToF MS analysis. The most active extract obtained using fresh roots and hexane/ethyl acetate (1:1) solvent mixture was able to significantly reduce content all examined mRNA. Qualitative chemical analysis reviled presence of 15 phenolic acids and their derivatives, 9 flavonoids and 10 glucosinolates in majority of examined extracts. It is significant to emphasize that the most active QSI extract did not contain a single one, out of ten dominant glucosinolates, which have undergone to hydrolysis yielding isothiocyanates and other sulphur-containing compounds responsible for QSI effects. Our results strongly indicate that even mild thermal treatment (40°C) of horseradish roots prior to extraction could lead to severe reduction or loss of QSI activity.

Antibiotic resistance represents a serious global health threat. Available antibiotics progressively lose efficacy against many bacterial pathogens. This phenomenon underscores the urgent need for alternative strategies to combat bacterial infections. Progress in understanding the regulation of bacterial pathogenicity has prompted researchers to explore potential antivirulence drugs as a promising alternative. Quorum quenching enzymes capable of degrading N-acyl homoserine lactones can impede bacterial virulence and biofilm formation by disrupting cell-to-cell communication. In this study, we employed in silico structural characterization of YtnP lactonase originating from Stenotrophomonas maltophilia 6960, utilizing various online software tools, including AlphaFold2, I-TASSER, PSIPRED, Phyre2, and SWISS-MODEL algorithms. The results obtained from these programs were compared to each other. The analysis revealed a 278 amino acid residue protein with a molecular weight of 31.02 kDa, predicted to be a transmembrane protein with an N-terminal extracellular domain and a C-terminal cytoplasmic domain, predominantly comprised of extracellular amino acid residues. Experimental validation demonstrated the quorum quenching activity of S. maltophilia 6960 towards exogenous AHLs, supporting the predicted role of YtnP lactonase in modulating quorum sensing of the surrounding bacteria. Furthermore, the observation of QQ activity in the crude extract and not in the cell-free supernatant of bacterial strain 6960 indicates that the YtnP lactonase is active within the bacterial cells. Secondary structure predictions revealed a balanced distribution of alpha-helices and beta-sheets, while tertiary structure predictions suggested a homodimeric configuration with four Zn2+ binding sites. These findings, which combine in silico predictions with experimental validation, provide a solid foundation for further exploration in the development of effective antivirulence therapeutics. Leverag

Polyphenols are well-known group of bioactive molecules distributed in various foods such as citruses and honey. They have been recognized mainly due to their antioxidant properties but recent insight into their antimicrobial spectrum implies potential novel application for this group. Polyphenols are able not only to block growth of pathogenic microorganisms, but they can also interfere with microbial ability to induce disease, microbial virulence. With the abundant studies conducted worldwide we now know about range of virulence factors that are necessary for disease induction and though we have new antimicrobial targets. One of the most studied virulence factors is microbial ability to form biofilms. We have recently studied rutin, flavonoid glycoside found in citruses. This molecule has the ability to block biofilm formation of Pseudomonas aeruginosa IBRS P001 and Staphylococcus aureus IBRS MRSA 011, strains that are resistant to antimicrobial therapeutics. In addition to reduction of biofilm biomass, rutin antibiofilm mechanisms also included reduction in cell viability, exopolysaccharide, and extracellular DNA levels. Moreover, moderate inhibition of bacterial adhesion to keratinocytes upon rutin treatment was observed. Rutin antivirulence mechanisms involved inhibition of P. aeruginosa protease, pyocyanin, rhamnolipid, and elastase production and the downregulation of the lasI, lasR, rhlI, rhlR, pqsA and mvfR genes. This research has proven wide antivirulence potential of rutin. Moreover, we have tested range of flavonoids as inhibitors of fungal virulence and resistance. Rutin, but also apigenin and apigetrin, have shown promising antibiofilm and anti-hyphal properties in several clinical Candida albicans strains examined, which was also confirmed on molecular level. Polyphenols have wide antivirulence capacity employing a range of mechanisms and might be used for the development of novel antimicrobial strategies. Intake of food polyphenols might have some h

Solving the problem of the antimicrobial resistance crisis is one of the primary challenges currently confronting the healthcare system. One of the most promising new strategies to combat antimicrobial resistance is the antivirulence therapy, based on silencing bacterial cell-to-cell communication (quorum quenching - QQ). QQ enzymes lactonases represent a diverse group of enzymes capable of inactivating signaling molecules of bacterial communication – N-acyl homoserine lactones (AHLs), resulting in alterations ofbacterial virulence. The numerous virulence factors and resistance to most conventional antibiotics have led to Pseudomonas aeruginosa being listed as one of the top-priority pathogens on the ESCAPE pathogen list, highlighting the urgent need for the development of new therapies to combat this pathogen. P. aeruginosauses cell-to-cell communication known as quorum sensing (QS) that allows bacteria to monitor their own population density via signal molecules and subsequently control bacterial pathogenesis. Our hypothesis was that bacterial pathogens which share the same ecological niche with P. aeruginosa during infection have developed a system to disrupt its QS system, in order to survive in polymicrobial communities alongside this successful pathogen. In our research we identified QQ enzymes lactonases originating from two Gramnegative bacterial pathogens Burkholderiacepacia and Stenotrophomonas maltophilia. The genes encoding for the enzymes were cloned and expressed in pQE30 expression vector. B. cepacia BCC4135 synthesizes two lactonases YtnP and Y2-aiiA, that have the different cellular localization, but also different substrate specificity, which could imply the difference in their biological roles. S. maltophilia 6960 YtnP lactonase has several advantageous biotechnological properties, such as high thermostability, activity in a wide pH range, and no cytotoxic microscopy analysis showed a strong effect of analyzed lactonases on preventing biofilm form

Antimicrobial resistance (AMR) and air pollution have been identified as one of the most serious threats to human health worldwide. The scarce data demonstrating their interdependence indicates a need to obtain evidence from a broader global area, especially from regions exposed to high air pollution. Considering that Serbia is a country struggling with excessive antibiotics use and misuse, a high percentage of multidrug-resistant bacterial isolates, and poor air quality, the Serbian capital Belgrade has been recognized as an interesting research model for the effects of air pollution on the airborne transmission of AMR. Hence, this abstract aims to present the concept of an ongoing project (AirPollRes) that points to the additional risk dimension of air pollution to human, animal, and environmental health. After optimization of air sampling and DNA extraction protocol, the air samples will be collected at nine locations in the Belgrade metropolitan area selected according to air pollution level during four seasons. The state-of-the-art shotgun metagenomic sequencing and bioinformatic analysis of obtained sequences will provide information about microbial community composition of airborne metagenomes. In addition, sequenced airborne metagenomes will be analyzed for the abundance and diversity of resistomes (antibiotic and biocide/metal resistance genes) and mobilomes using several databases and tools. Correlation analyses will offer us insight into the effect of air pollution and seasonal variations on abundance and diversity of airborne pathogens, resistome and mobilome in the Belgrade metropolitan area. In-depth approach of the AirPollRes project will provide the first insights into intersection of AMR and air pollution in the Belgrade metropolitan area, which is highly vulnerable to these health threats. As the AirPollRes is a pioneering project in this field, the expected shortterm impact is the introduction of routine monitoring of pathogenic microbes and resis

Pseudomonas aeruginosa is a Gram-negative pathogen responsible for frequent hospital-acquired infections of the bloodstream, the respiratory tract, and the urinary tract. Quorum quenching enzymes are recognized as an alternative antivirulence approach targeting pathogenic bacteria. The efficacy of YtnP lactonase in reducing the virulence of P. aeruginosa MMA83 in vivo using Caenorhabditis elegans as a model system was investigated. The recombinant YtnP lactonase exhibits no cytotoxicity, demonstrated by its lack of harmful effects on both the immortalized human HaCaT cell line and two strains of C. elegans (AU37 and N2 wild-type). In a toxin-mediated killing liquid assay, the survival rates of C. elegans AU37 mutant and N2 wildtype strains infected with the clinical isolate P. aeruginosa MMA83 significantly increased when pre-treated with YtnP lactonase, compared to untreated controls. Considering that virulence factors expression is regulated by quorum sensing (QS) signaling it is hypothesized that YtnP lactonase prolongs the life span of C. elegans by downregulating the QS and expression of virulence factors of MMA83. The protective effects of YtnP lactonase against MMA83-induced pathogenicity in C. elegans, coupled with its absence of cytotoxicity, position YtnP lactonase as a promising prophylactic agent with antivirulence properties.

Pseudomonas aeruginosa has been recognized as a priority pathogen by World Health Organization, due to the emergence of multidrug-resistant (MDR) strains. Thus, new treatment options such as antivirulence strategy is urgently needed. This strategy is based on the disruption of quorum sensing (QS) activity of this pathogen. The focus of this research was to explore the anti-QS activity of four selected medicinal mushrooms (Lentinula edodes, Cantharellus cibarius, Trametes versicolor and Pleurotus ostreatus) extracts on MDR clinical isolate P. aeruginosa MMA83. Another aim was to check their cytotoxicity on Caenorhabditis elegans AU37 (glp-4(bn2) I; sec-1(km4). Among three types of mushroom extracts - hot water polysaccharide extracts (WPE), hot alkali polysaccharide extracts (APE) and methanol extracts (Met), APE extracts downregulated all tested QS and virulence factors genes of P. aeruginosa MMA83. The most prominent effect was observed for C. cibarius APE extract, lowering expression from 2-fold (for lasI gene) to 20-fold for lasB gene. Extracts didn’t show cytotoxic effect on C. elegans. The efficacy of APE extracts in lowering the expression of QS and virulence factors genes of P. aeruginosa MMA83 indicate that these extracts can reduce pathogenicity of P. aeruginosa. Also, they possess one of the desirable biotechnology features – the absence of cytotoxicity. Anti-QS and antivirulence effect of APE extracts on P. aeruginosa envisages these extracts as the promising therapeutic candidates for the development of next-generation antivirulence agents.

Biofilm-associated infections are the main cause of biomaterial implant failure today. The increasing prevalence of antibiotic-resistant pathogens often results in the only solution of implant movement, with serious consequences for patients. Recently, various antimicrobial agents have been recognized as a promising strategy to prevent biofilm formation on implant surfaces. Quorum sensing (QS) plays a central role in the control of bacterial virulence and biofilm formation. The use of quorum quenching (QQ) enzymes to target QS is therefore a promising innovative approach for the development of enzyme-based antivirulence therapeutics, which represent a potential solution to combat infections caused by multidrug-resistant pathogens. This study aimed to characterize the novel YtnP lactonase from the clinical isolate Stenotrophomonas maltophilia 6960 and to investigate its potential to combat the virulence of multidrug-resistant (MDR) Pseudomonas aeruginosa MMA83.

Introduction: Brevibacillus laterosporus is a promising microbiological agent that can be used to prevent and control destructive diseases affecting honey bee colonies. In the presentstudy, the short-term effect of the B. laterosporus BGSP11 bee diet on microbiota and mycobiota was investigated. Methods: The honey bee diet was supplemented with spores of B. laterosporus BGSP11 at a concentration of 1×108 CFU/mL in sucrose solution. Metabarcoding analysis of the bee microbial community profile was performed based on 16S RNA (bacteriobiota) and Internally Transcribes Spacer (ITS) region (mycobiota) obtained using MiSeq Illumina sequencing. The QIIME2 v2021.4 pipeline was used to analyze the obtained amplicon data library. Results: The results show that the BGSP11 bee diet slightly altered the bee microbiota and did not lead to potentially harmful changes in the bacterial microbiota. Moreover, it can potentially induce positive changes, mainly reflected in the reduction of opportunistic bacteria. On the other hand, the treatment had a greater effect on mycobiota. However, the changesin the bee mycobiome caused by the treatment cannot be considered a priori as beneficial or harmful,since the interaction between the bee and its mycobiome is not sufficiently studied. The observed positive changes in the bee mycobiome are mainly reflected in the reduction of phytopathogenic fungi that may affect the organoleptic and techno-functional properties of honey. Conclusion: This pilot study suggests that the introduction of BGSP11 in beekeeping practice as a biological agent could be considered due to no harmful effects observed on the microbiota of bees.

Thermal springs represent a habitat with extreme conditions that harbor a unique microbial community adapted to thrive in this environment. In addition to the geothermal springs in Iceland, the thermal springs of Vranjska Banja are considered the hottest in Europe with a water temperature of 63-95°C. Due to global warming and climate change, there is a growing need for knowledge about the biodiversity of extreme natural habitats. Besides the exceptional importance of studying extremophilic microorganisms, the difficulty in their cultivation limits the expanding necessity of research in this field. This study provides information about the microbial community structure and physicochemical characteristics of the thermal spring of Vranjska Banja. To determine and monitor the microbiota diversity of the Vranjska Banja hot spring, for the first time, comprehensive culture-independent metagenomic analysis in parallel with a culture-dependent approach was applied. The culture-independent composition of bacterial communities of the thermal water was investigated using MiSeq-Illumina technology and analyzed by the computing environment QIIME2 v2021. The applied cultivation approach resulted in the isolation of 17 strains belonging to genera Bacillus, Anoxybacillus, Hydrogenophilus, and Geobacillus, based on 16S rRNA sequencing and whole genome sequencing of five representative strains has been performed. The complete DNA was sequenced using Illumina HiSeq from the MicrobesNG service. Genomic characterization and OrthoANI analysis have shown that two of them are candidates for novel species. Products of extremophilic microorganisms adapted to harsh conditions have great potential to be used for biotechnological research and industrial application. Results of BAGEL4 and AntiSMASH showed that the sequenced strains from Vranjska Banja hot spring have the potential to produce thermostable enzymes (proteases, lipases, amylases, phytase, chitinase, and glucanase) and various antimi

In the current study, honey bees’ diet was supplemented with spores of Brevibacillus laterosporus BGSP11 at concentration of 108 CFU/ml in sucrose solution and its short-term effects on their micro- and mycobiota have been analyzed using Illumina MiSeq sequencing. Obtained results indicate that this treatment does not lead to potentially harmful changes in the bacterial microbiome of worker bees, slightly affecting the composition of core microbiota. Moreover, several potentially beneficial changes have been observed. The treatment has led to a significant increase in the abundance of Snodgrassella alvi, and species from Lactobacillus and Bifidobacterium genera which play important roles in protection against several honey bee pathogens. Simultaneously, B. laterosporus enriched diet have led to almost complete eradication of Enterobacteriaceae family, the taxon that contains several putative pathogen species. On the other hand, the treatment affected mycobiota more profoundly, which was expected considering the greater instability compared to microbiota. Although the observed changes in honey bee mycobiome cannot be considered a priori beneficial or harmful, since the interaction between the bee and its mycobiome has not been sufficiently studied, certain beneficial consequences of the treatment have been observed. They are primarily reflected in the reduction of phytopathogenic fungi that can affect the organoleptic and techno-functional characteristics of honey. In addition, before introducing B. laterosporus in beekeeping practice as a biological agent for pathogen control it is necessary to perform more thorough studies of the impact on the honey bee microbiome, immune system, physiology and economic characteristics of honey bee colonies.

Klebsiella pneumoniae is a global health threat and bacteriophages are a potential solution in combating pandrug-resistant K. pneumoniae infections. Two lytic phages, LASTA and SJM3, active against several pandrug-resistant, nosocomial strains of K. pneumoniae were isolated and characterized. Their host range is narrow and latent period is particularly long; however, their lysogenic nature was refuted using both bioinformatic and experimental approaches. Genome sequence analysis clustered them with only two other phages into the new genus Lastavirus. Genomes of LASTA and SJM3 differ in only 13 base pairs, mainly located in tail fiber genes. Individual phages, as well as their cocktail, demonstrated significant bacterial reduction capacity in a time-dependent manner, yielding up to 4 log reduction against planktonic, and up to 2.59 log on biofilm-embedded, cells. Bacteria emerging from the contact with the phages developed resistance and achieved numbers comparable to the growth control after 24 h. The resistance to the phage seems to be of a transient nature and varies significantly between the two phages, as resistance to LASTA remained constant while resensitization to SJM3 was more prominent. Albeit with very few differences, SJM3 performed better than LASTA overall; however, more investigation is needed in order to consider them for therapeutic application.

Lactic acid bacterium Lactococcus lactis BGBU1-4 produces 43 amino acids (aa) long bacteriocin, lactolisterin BU (LBU), a 5.161 kDa peptide with potent antibacterial activity against many Gram-positive pathogens. In addition, BGBU1-4 produces an additional unknown product of 3.642 kDa with antibacterial activity. Here, we determined that the significant amount of naturally produced LBU breaks down to create a 3.642 kDa truncated form of LBU bacteriocin consisting of 31 N-terminal aa (LBU1-31) that exhibits 12.5% the antibacterial activity of the full-length LBU. We showed that chemically synthesized LBU is stable and 50% less active than native LBU, and so we used the synthetic peptides of LBU and its variants to further study their activities and antibacterial potential. Deletion analysis of LBU revealed that the 24 N-terminal aa of LBU (LBU1-24) are responsible for antibacterial activity, while downstream aa (25–43) determine the species-specific effectiveness of LBU. Although LBU1-31 contains aa 1–24, the truncation at position 31 is predicted to change the structure within aa 15–31 and might impact on antibacterial activity. Intriguingly, whole genome sequencing and genome mining established that BGBU1-4 is abundant in genes that encode potential antibacterials, but produces LBU and its breakdown product LBU1-31 exclusively.

The hot spring Vranjska Banja is the hottest spring on the Balkan Peninsula with a water temperature of 63–95 °C and a pH value of 7.1, in situ. According to the physicochemical analysis, Vranjska Banja hot spring belongs to the bicarbonated and sulfated hyperthermal waters. The structures of microbial community of this geothermal spring are still largely unexplored. In order to determine and monitor the diversity of microbiota of the Vranjska Banja hot spring, a comprehensive culture-independent metagenomic analysis was conducted in parallel with a culture-dependent approach for the first time. Microbial profiling using amplicon sequencing analysis revealed the presence of phylogenetically novel taxa, ranging from species to phyla. Cultivation-based methods resulted in the isolation of 17 strains belonging to the genera Anoxybacillus, Bacillus, Geobacillus, and Hydrogenophillus. Whole-genome sequencing of five representative strains was then performed. The genomic characterization and OrthoANI analysis revealed that the Vranjska Banja hot spring harbors phylogenetically novel species of the genus Anoxybacillus, proving its uniqueness. Moreover, these isolates contain stress response genes that enable them to survive in the harsh conditions of the hot springs. The results of the in silico analysis show that most of the sequenced strains have the potential to produce thermostable enzymes (proteases, lipases, amylases, phytase, chitinase, and glucanase) and various antimicrobial molecules that can be of great importance for industrial, agricultural, and biotechnological applications. Finally, this study provides a basis for further research and understanding of the metabolic potential of these microorganisms.

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.

Signal transduction systems are the key players of bacterial adaptation and survival. The orthodox two-component signal transduction systems perceive diverse environmental stimuli and their regulatory response leads to cellular changes. Although rarely described, the unorthodox three-component systems are also implemented in the regulation of major bacterial behavior such as the virulence of clinically relevant pathogen P. aeruginosa. Previously, we described a novel three-component system in P. capeferrum WCS358 (RclSAR) where the sensor kinase RclS stimulates the intI1 transcription in stationary growth phase. In this study, using rclS knock-out mutant, we identified RclSAR regulon in P. capeferrum WCS358. The RNA sequencing revealed that activity of RclSAR signal transduction system is growth phase dependent with more pronounced regulatory potential in early stages of growth. Transcriptional analysis emphasized the role of RclSAR in global regulation and indicated the involvement of this system in regulation of diverse cellular activities such as RNA binding and metabolic and biocontrol processes. Importantly, phenotypic comparison of WCS358 wild type and Delta rclS mutant showed that RclS sensor kinase contributes to modulation of antibiotic resistance, production of AHLs and siderophore as well as host cell adherence and cytotoxicity. Finally, we proposed the improved model of interplay between RclSAR, RpoS and LasIR regulatory systems in P. capeferrum WCS358.

Owing to the emerging resistance to antimicrobials in Salmonella Kentucky isolates around the globe, the genomic comparison of all the registered multidrug-resistant Salmonella Kentucky isolates in Serbia (five from humans, one from turkey flock, and one from meat) was done. Most of the isolates were isolated from patients returning from Egypt or Tunisia or originated from imported turkey flock and turkey meat. The comparative analysis of resistance and virulence genes was done. All isolates belonged to sequence type-ST198 and were resistant to ciprofloxacin (Cip). The resistance to Cip was mediated by target mutations of the gyrA and parC genes, which encode topoisomerase I and II, respectively. Multidrug-resistant phenotype to aminoglycosides, beta-lactam antibiotics, sulfonamides, and tetracyclines was detected in five isolates. However, none of the isolates was pan-resistant to antimicrobials. The number of single nucleotide polymorphisms between isolates varied from 8 to 43 and phylogenomics revealed the genetic proximity of the human isolate 10475/11 and the turkey meat isolate 5264/14, indicating a possible meat-to-human transfer. All isolates belonged to the main Salmonella Kentucky MDR lineage, carrying the Salmonella genomic island 1 (SGI1-K) subtype. The SGI1-K of Serbian isolates showed mosaicism attributed to rapid intraclonal evolution. Many virulence factors were detected in all the isolates, including SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-9, and C63PI. Although Salmonella Kentucky has rarely been isolated from humans, food, and animals in Serbia, further surveillance is needed to diminish the risk of the spreading of resistant clones and their meat-to-human transmission.

The rising incidence of antibiotic resistant microorganisms urges novel antimicrobials development with polyphenols as appealing potential therapeutics. We aimed to reveal the most promising polyphenols among hesperetin, hesperidin, naringenin, naringin, taxifolin, rutin, isoquercitrin, morin, chlorogenic acid, ferulic acid, p-coumaric acid, and gallic acid based on antimicrobial capacity, antibiofilm potential, and lack of cytotoxicity towards HaCaT, and to further test its antivirulence mechanisms. Although the majority of studied polyphenols were able to inhibit bacterial growth and biofilm formation, the most promising activities were observed for rutin. Further investigation proved rutin's ability to prevent/eradicate Pseudomonas aeruginosa and MRSA urinary catheter biofilms. Besides reduction of biofilm biomass, rutin antibiofilm mechanisms included reduction of cell viability, exopolysaccharide, and extracellular DNA levels. Moderate reduction of bacterial adhesion to human keratinocytes upon treatment was observed. Rutin antivirulence mechanisms included an impact on P. aeruginosa protease, pyocyanin, rhamnolipid, and elastase production and the downregulation of the lasI, lasR, rhlI, rhlR, pqsA and mvfR genes. Rutin also interfered with membrane permeability. Polyphenols could repress antibiotic resistant bacteria. Rutin has shown wide antimicrobial and antibiofilm capacity employing a range of mechanisms that might be used for the development of novel antimicrobials.

Among them, bacterial and fungal pathogens Paenibacillus larvae, Melissococcus pluton, Ascosphera apis andNosema ceranae play a major impact on honey bees colonies. Thus, developing alternative prophylactic andcurative strategies are urgently needed. The use of probiotic bacteria in honey bee supplemental feeding istherefore promising to treat or prevent diseases. Brevibacillus laterosporus, Gram-positive endospore formingbacilli, is recognised as one of the promising antibacterial and antifungal agents producer.The aim of this study was to examine the short-therm effects of B. laterosporus supplemented diet on workerhoney bee microbiome.Dry spores of B. laterosporus strain BGSP11 have been administrated through a sugar syrup diet to tencolonies and a representative specimen of worker honey bees was taken before the start of the treatmentand immediately after the syrup was consumed. The microbial diversity was assessed before and after thetreatment using Illumina MiSeq sequencing platforms (ID Genomics service, Seattle, WA, USA). 16s rRNAsequencing for bacterial community profiling and fungal Internally Transcribes Spacer for mycological taxaprofiling were used. The next-generation microbiome bioinformatics platform QIIME2 v 2021.4 was used forfiltering and denoising obtained sequences, calculation of diversity metrics and taxonomy assignment. Thefeature classifier was trained using the Greengenes v 13_8 for bacterial taxa and fungal UNITE database v 8.3.The results obtained in this study indicated statisticaly significant alfa diversity between control and experimentalgroup honey bee microbiota composition. The diversity abundance was higher in control comparingto the group treated with B. laterosporus strain BGSP11 spores. There was no significant diference in Bray-Curtis distance among two groups of analysed samples. Regarding to mycological abundance, compositionwas completely different between two groups; control group had Claviceps as predominant genus, while

Pseudomonas aeruginosa jedan je od najznačajnijih uzročnika unutarbolničkih infekcija čiji je terapijski tretman konvencionalnim antibioticima sve češće neefikasan usled rezistencije na antibiotike. Inovativni vidovi kontrole infekcija, poput utišavanja međućelijske komunikacije bakterija, a time i onemogućavanja virulencije i inhibicije patogenog fenotipa su stoga od izuzetnog značaja. U ovom radu biće predstavljena istraživanja koja su bazirana na prirodnom svojstvu bakterija koje dele ekološke niše da sarađuju, ali i kompetiraju, na osnovu čega su analizirane Delftia tsuruhatensis i Burkholderia cepacia koje tokom infekcija kolokalizuju sa P. aeruginosa. Pokazano je da D. tsuruhatensis 11304 produkuje C18-HSL koji inhibira virulenciju P. aeruginosa i rekonstituiše osetljivost na antibiotike, a takođe je po prvi put u literaturi opisano prisustvo dihidroksi- C18-HSL u biološkim uzorcima. Opisane su i laktonaze vrste B. cepacia BCC4135 koje degraduju autoinducere komunikacije P. aeruginosa i inhibiraju ekspresiju faktora virulencije. Utvrđena je njihova supstratna specifičnost i ukazano na različitu biološku funkciju u zavisnosti od lokalizacije., Pseudomonas aeruginosa is a leading cause of nosocomial infections, whose therapeutic treatment with conventional antibiotics is increasingly ineffective due to antibiotic resistance. Inovative approaches of infection control, such as silencing the bacterial quorum sensing system and thus virulence and pathogenic phenotype inhibition are of great importance. In this study, there will be presented research based on natural feature of bacteria that share the same ecological niche to coordinate, but also to compete, based on which Delftia tsuruhatensis and Burkholderia cepacia that colocalize with P. aeruginosa during infections were analysed. D. tsuruhatensis 11304 has been shown to produce C18-HSL which inhibits P. aeruginosa virulence and reconstitutes antibiotic susceptibility, and the presence of dihydroxy-C18-HSL in biological samples has also been described for the first time in the literature. B. cepacia BCC4135 lactonases that degrade autoinducers of P. aeruginosa quorum sensing system and inhibit virulence factor expression have also been reported. Their substrate specificity was determined and different biological function depending on their localization was indicated.

Nemogućnost kontrole pojave i širenja rezistencije na antibiotike usmerila je istraživanja u poslednje dve dekade ka iznalaženju novih terapeutskih opcija, sa ciljem tretmana infekcija izazvanih patogenim bakterijama rezistentnim na veći broj kliniĉki znaĉajnih antibiotika. Uzevši u obzir ĉinjenicu da je antibiotska rezistencija kompleksan, multifaktorijalni fenomen, rešenje ovog problema ukljuĉuje niz pristupa usmerenih na kontrolu faktora koji olakšavaju nastanak i širenje rezistencije. Jedan od tih pristupa sastoji se u razvoju novih terapeutika koji bi delovali mehanizmima razliĉitim od trenutno dostupnih antibiotika. U tom pogledu, antivirulentna terapija zamišljena je kao obećavajuća alternativa sa ciljem kontrole virulencije specifiĉne za odreĊene patogene, bez vršenja snažnog selektivnog pritiska na bakterijske ćelije. Imajući u vidu da je Pseudomonas aeruginosa jedan od vodećih uzroĉnika unutarbolniĉkih infekcija širom sveta, leĉenje infekcija izazvanih ovim patogenom predstavlja veliki terapeutski izazov. P. aeruginosa svoj patogeni potencijal ostvaruje zahvaljujući mnogobrojnim uroĊenim, steĉenim i adaptivnim mehanizmima rezistencije. Pored toga, posedovanje sistema meĊućelijske komunikacije (eng. quorum sensing, QS) ovom patogenu omogućava fleksibilnost u regulaciji ekspresije gena ukljuĉenih u virulenciju, formiranje biofilma, produkciju sekundarnih metabolita i faktora koji imaju ulogu u zaštiti od imunskog sistema domaćina. Stoga bi primena antivirulentne terapije zasnovane na utišavanju meĊućelijske komunikacije bakterija mogla poslužiti kao obećavajuće oruĊe u kontroli infekcija izazvanih predstavnicima P. aeruginosa za koje ne postoji adekvatna terapija trenutno dostupnim antibioticima. U skladu sa time, predmet prouĉavanja ove teze bila je potraga za novim utišivaĉima meĊućelijske komunikacije bakterija (eng. quorum quenching, QQ) produkovanih od strane kliniĉkih izolata koji tokom infekcija dele istu ekološku nišu sa P. aeruginosa. Analizirana je, finding novel therapeutic options to combat multidrug-resistant pathogenic bacteria during the last two decades. Given the fact that antimicrobial resistance is a complex, multifactorial phenomenon, the solution to this problem comprises a range of approaches focused on monitoring the factors that facilitate the emergence and spread of resistance. One of proposed strategies consists of developing novel therapeutics that operate under different principles to the currently available antibiotics. In this respect, antivirulence therapy has been conceived as a promising alternative to control virulence in a pathogen-specific manner, without exerting strong selective pressure on the bacterial cells. Having in mind that Pseudomonas aeruginosa has been considered as a leading cause of nosocomial infections worldwide, the treatment of infections caused by this pathogen represents a major therapeutic challenge. The pathogenic potential of P. aeruginosa has been accomplished due to a numerous innate, acquired, and adaptive resistance mechanisms. In addition, the presence of cell-to-cell communication system (quorum sensing, QS) allows this pathogen the flexibility in the regulation of virulence gene expression responsible for biofilm development, production of secondary metabolites, and immuneevasive factors. Therefore, the use of antivirulence therapy based on the silencing of bacterial communication could serve as a promising tool in the control of infections caused by P. aeruginosa for which there is no adequate therapy with currently available antibiotics. Accordingly, the subject of this thesis was discovery and characterization of novel quenchers of bacterial cell-to-cell communication produced by clinical isolates that share the same ecological niche with P. aeruginosa during infections. A collection of Gram-negative clinical isolates from the Laboratory for Molecular Microbiology Institute of Molecular Genetics and Genetic Engineering University of Belgrade was analyze

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.

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.

The gene cluster responsible for the production of the aureocin A53-like bacteriocin, lactolisterin BU, is located on plasmid pBU6 in Lactococcus lactis subsp. lactis BGBU1-4. Heterologous expression of pBU6 confirmed that production and limited immunity to lactolisterin BU were provided by the plasmid. Comparative analysis of aureocin A53-like operons revealed that the structural genes shared a low level of identity, while other genes were without homology, indicating a different origin. Subcloning and expression of genes located downstream of the structural gene, IliBU, revealed that the lactolisterin BU cluster consists of four genes: the structural gene IliBU, the abcT gene encoding an ABC transporter, the accL gene encoding an accessory protein and the immL gene which provides limited immunity to lactolisterin BU. Reverse transcription analysis revealed that all genes were transcribed as one polycistronic mRNA. Attempts to split the lactolisterin BU operon, even when both parts were under control of the PlliBU promoter, were unsuccessful indicating that expression of lactolisterin BU is probably precisely regulated at the translational level by translational coupling and is possible only when all genes of the operon are in cis constellation. Two rho-independent transcription terminators were detected in the lactolisterin BU operon: the first in the intergenic region of the IliBU and abcT genes and the second at the end of operon. Deletion of the second transcription terminator did not influence production of the bacteriocin in lactococci.

Acinetobacter baumannii has been recognized as one of the most challeging pathogens in clinical settings worldwide. Outer membrane porins play a significant role in Acinetobacter antibiotic resistance and virulence. A. baumannii carbapenem resistance and virulence factor porin Omp33-36 was the subject of this study. We investigated the omp33-36 gene transcriptional response in the growth phase, its response to carbapenems, and the effect of contact with host cells. Additionally, the cytotoxic effect of A. baumannii towards keratinocytes was assessed, as well as correlation between omp33-36 gene transcription and cytotoxicity. Further, Acinetobacter spp. Omp33-36 was classified and its characteristics relevant for vaccine candidature were determined. The level of the omp33-36 gene transcription varied between growth phases, but a common pattern could not be established among different strains. Treatment with subinhibitory concentrations of carbapenems decreased, while contact with keratinocytes increased omp33-36 expression in the analysed A. baumannii strains. Variations in omp33-36 mRNA levels did not correlate with cytotoxicity levels. Decrease of omp33-36 mRNA during treatment with subinhibitory concentrations of carbapenems, indicated the importance of transcriptional changes in reversible resistance to carbapenems due to the absence of Omp33-36. The transcription of omp33-36 increased after contact with keratinocytes, indicating the important role of de novo transcription during the initial phase of A. baumannii infection. Primary structural analysis of Acinetobacter spp. Omp33-36 revealed three distinct groups (among four A. baumannii variants). Although we have shown that Omp33-36 was highly polymorphic, we propose a potential antigen (PLAEAAFL motif) for vaccine development. According to PROVEAN analysis, the highly polymorphic structure of Omp33-36 porin should not influence its function significantly.

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.

Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 produces a novel bacteriocin, lactolisterin BU, with strong antimicrobial activity against many species of Gram-positive bacteria, including important food spoilage and foodborne pathogens, such as Listeria monocytogenes, Staphylococcus aureus, Bacillus spp., and streptococci. Lactolisterin BU was extracted from the cell surface of BGBU1-4 by 2-propanol and purified to homogeneity by C18 solid-phase extraction and reversed-phase high-performance liquid chromatography. The molecular mass of the purified lactolisterin BU was 5,160.94 Da, and an internal fragment, AVSWAWQH, as determined by N-terminal sequencing, showed low-level similarity to existing antimicrobial peptides. Curing and transformation experiments revealed the presence of a corresponding bacteriocin operon on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4. Analysis of the bacteriocin operon revealed a leaderless bacteriocin of 43 amino acids that exhibited similarity to bacteriocin BHT-B (63%) from Streptococcus ratti, a bacteriocin with analogy to aureocin A. IMPORTANCE Lactolisterin BU, a broad-spectrum leaderless bacteriocin produced by L. lactis subsp. lactis bv. diacetylactis BGBU1-4, expresses strong antimicrobial activity against food spoilage and foodborne pathogens, such as Listeria monocytogenes, Staphylococcus aureus, Bacillus spp., and streptococci. Lactolisterin BU showed the highest similarity to aureocin-like bacteriocins produced by different bacteria. The operon for synthesis is located on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4, indicating possible horizontal transfer among producers.