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).... more
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 resist...
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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... more
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 immuno...
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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... more
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 μg 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 μg ml−1) used in combination with SeNPs (0.5 μg ml−1) was able to reduce initial inoculum during the first 4 h of incubation, in contrast to colistin (0.5, 1 or 2 μg ml−1) alone. Conclusions These findings propose colistin/SeNPs combination as a new option to fight PDR Ac. baumannii, the therapeutic possibilities of wh...
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Pseudomonas aeruginosa is one of the most common opportunistic pathogens that use quorum sensing (QS) system to regulate virulence factors expression and biofilm development. Delftia sp. 11304 was selected among 663 Gram-negative clinical... more
Pseudomonas aeruginosa is one of the most common opportunistic pathogens that use quorum sensing (QS) system to regulate virulence factors expression and biofilm development. Delftia sp. 11304 was selected among 663 Gram-negative clinical isolates based on its QS inhibitory activity against P. aeruginosa MMA83 clinical isolate. Whole genome sequencing identified this isolate as D. tsuruhatensis and revealed genetic armamentarium of virulence factors and antibiotic resistance determinants. Ethyl acetate extract of D. tsuruhatensis 11304 culture supernatant (QSI extract) prevented biofilm formation of P. aeruginosa MMA83, but was unable to cause biofilm decomposition. QSI extract showed a synergistic effect in combination with meropenem and gentamycin, against P. aeruginosa MMA83. A dose-dependent reduction of the virulence factors: elastase, rhamnolipid and pyocyanin production by P. aeruginosa MMA83 and significant downregulation of lasI, lasR, rhlI, rhlR, pqs and mvfR expression we...
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Ever since the development of the first vaccine, vaccination has had the great impact on global health, leading to the decrease in the burden of numerous infectious diseases. However, there is a constant need to improve existing vaccines... more
Ever since the development of the first vaccine, vaccination has had the great impact on global health, leading to the decrease in the burden of numerous infectious diseases. However, there is a constant need to improve existing vaccines and develop new vaccination strategies and vaccine platforms that induce a broader immune response compared to traditional vaccines. Modern vaccines tend to rely on certain nanotechnology platforms but are still expected to be readily available and easy for large-scale manufacturing and to induce a durable immune response. In this review, we present an overview of the most promising nanoadjuvants and nanoparticulate delivery systems and discuss their benefits from tehchnological and immunological standpoints as well as their objective drawbacks and possible side effects. The presented nano alums, silica and clay nanoparticles, nanoemulsions, adenoviral-vectored systems, adeno-associated viral vectors, vesicular stomatitis viral vectors, lentiviral v...
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<p>IC50 values, genotyping results and CarO variants for <i>A</i>. <i>baumannii</i> isolates from Serbia.</p
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<p>Minimal inhibitory concentrations (MICs).</p
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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... more
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 com-plex 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...
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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... more
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. Sin...
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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... more
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 38 clusters of attC sites lacking integron-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 P...
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Restriction enzymes are the main defence system against foreign DNA, in charge of preserving genome integrity. BGTRK10-1 expresses I Type II restriction-modification enzyme, whose activity is similar to that shown for RI; I... more
Restriction enzymes are the main defence system against foreign DNA, in charge of preserving genome integrity. BGTRK10-1 expresses I Type II restriction-modification enzyme, whose activity is similar to that shown for RI; I methyltransferase protects DNA from RI cleavage. The gene encoding I endonuclease was cloned and overexpressed in . Purified enzyme showed the highest specific activity at lower temperatures (between 13°C and 37°C) and was stable after storage at -20°C in 50% glycerol. The concentration of monovalent ions in the reaction buffer required for optimal activity of I restriction enzyme was 100 mM or higher. The recognition and cleavage sequence for I restriction enzyme was determined as 5'-G/AATTC-3', indicating that I restriction enzyme is an isoschizomer of RI. In the reaction buffer with a lower salt concentration, I exhibits star activity and specifically recognizes and cuts another alternative sequence 5'-A/AATTC-3', leaving the same sticky ends o...
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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.... more
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). 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. 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). ...
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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 Lis Lactococcus lactis. L. lactis strains are of great importance in the... more
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 Lis 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 includ?ing 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 an...
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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.... more
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 m...
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New Delhi metallo-beta-lactamase-1 (NDM-1) will soon become the most commonly isolated and distributed metallo-beta-lactamase worldwide due to its rapid international dissemination and its ability to be expressed by numerous Gram-negative... more
New Delhi metallo-beta-lactamase-1 (NDM-1) will soon become the most commonly isolated and distributed metallo-beta-lactamase worldwide due to its rapid international dissemination and its ability to be expressed by numerous Gram-negative pathogens. NDM-positive bacteria pose a significant public health threat in the Indian subcontinent and the Balkans, which have been designated as endemic regions. Our study was focused on urban rivers, a lake and springheads as a potential source of NDM-1-producing strains in Serbia, but also as a source of other metallo-beta-lactamases and extended-spectrum beta-lactamase (ESBL) producing bacteria. A total of 69 beta-lactam resistant isolates, belonging to 12 bacterial genera, were collected from 8 out of 10 different locations in Belgrade, of which the most were from a popular recreational site, Ada Ciganlija Lake. Phenotypic tests revealed 7 (10.14%) ESBL-producing isolates and 39 (56.52%) isolates resistant to imipenem, of which 32 were positi...
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Functional characterization of the multidrug resistance CmbT transporter was performed in Lactococcus lactis. The cmbT gene is predicted to encode an efflux protein homologous to the multidrug resistance major facilitator superfamily. The... more
Functional characterization of the multidrug resistance CmbT transporter was performed in Lactococcus lactis. The cmbT gene is predicted to encode an efflux protein homologous to the multidrug resistance major facilitator superfamily. The cmbT gene (1377 bp) was cloned and overexpressed in L. lactis NZ9000. Results from cell growth studies revealed that the CmbT protein has an effect on host cell resistance to lincomycin, cholate, sulbactam, ethidium bromide, Hoechst 33342, sulfadiazine, streptomycin, rifampicin, puromycin and sulfametoxazole. Moreover, in vivo transport assays showed that overexpressed CmbT-mediated extrusion of ethidium bromide and Hoechst 33342 was higher than in the control L. lactis NZ9000 strain. CmbT-mediated extrusion of Hoechst 33342 was inhibited by the ionophores nigericin and valinomycin known to dissipate proton motive force. This indicates that CmbT-mediated extrusion is based on a drug-proton antiport mechanism. Taking together results obtained in thi...
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Adhesion of bacteria to mucosal surfaces and epithelial cells is one of the key features for the selection of probiotics. In this study, we assessed the adhesion property of Lactococcus lactis subsp. lactis BGKP1 based on its strong... more
Adhesion of bacteria to mucosal surfaces and epithelial cells is one of the key features for the selection of probiotics. In this study, we assessed the adhesion property of Lactococcus lactis subsp. lactis BGKP1 based on its strong autoaggregation phenotype and the presence of the mucin binding protein (MbpL). Genes involved in aggregation ( aggL ) and possible interaction with mucin ( mbpL ), present on the same plasmid pKP1, were previously separately cloned in the plasmid pAZIL. In vivo and in vitro experiments revealed potentially different physiological roles of these two proteins in the process of adherence to the intestine during the passage of the strain through the gastrointestinal tract. We correlated the in vitro and in vivo aggregation of the BGKP1-20 carrying plasmid with aggL to binding to the colonic mucus through nonspecific hydrophobic interactions. The expression of AggL on the bacterial cell surface significantly increased the hydrophobicity of the strain. On the...
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New Delhi metallo-β-lactamase producing Pseudomonas aeruginosa isolates are of special interest since P. aeruginosa is a major cause of nosocomial infections, the treatment of which could now be jeopardized, especially in developing... more
New Delhi metallo-β-lactamase producing Pseudomonas aeruginosa isolates are of special interest since P. aeruginosa is a major cause of nosocomial infections, the treatment of which could now be jeopardized, especially in developing countries. Six additional NDM-1 positive P. aeruginosa clinical isolates belonging to two different genotypes were shown to be plasmid-free. PFGE-hybridization experiments revealed the chromosomal location of the blaNDM-1 gene. Restriction analysis and hybridization revealed that two copies of the blaNDM-1 gene are present in the genomes of all tested isolates, as in previously characterized P. aeruginosa MMA83. Moreover, it was shown that increasing imipenem concentration did not have the effect on copy number of the blaNDM-1 gene in the genome of P. aeruginosa MMA83.
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<p>The dendrogram was constructed using SPSS software. Letters A, B, C and D indicate different pulsotypes, while I and II designate two major clusters.</p
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SDS-PAGE gel electrophoresis of proteins precipitated from supernatant of <i>Br</i>. <i>laterosporus</i> BGSP7, BGSP9, BGSP11 and BGSP12 strains on 15% acrylamide gel stained with Coomassie Brilliant Blue (A); gel with the same samples overlayed with LA-soft agar containing <i>S</i>. <i>aureus</i...more
<p>SDS-PAGE gel electrophoresis of proteins precipitated from supernatant of <i>Br</i>. <i>laterosporus</i> BGSP7, BGSP9, BGSP11 and BGSP12 strains on 15% acrylamide gel stained with Coomassie Brilliant Blue... more
<p>SDS-PAGE gel electrophoresis of proteins precipitated from supernatant of <i>Br</i>. <i>laterosporus</i> BGSP7, BGSP9, BGSP11 and BGSP12 strains on 15% acrylamide gel stained with Coomassie Brilliant Blue (A); gel with the same samples overlayed with LA-soft agar containing <i>S</i>. <i>aureus</i> ATCC25923 after incubation overnight at 37°C (B).</p
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<p>(A) Shannon variability of CarO sequences within groups I, II, and III. The height of each bar represents the entropy of an amino acid residue at a given position. The signal CarO peptide sequence is included in this analysis.... more
<p>(A) Shannon variability of CarO sequences within groups I, II, and III. The height of each bar represents the entropy of an amino acid residue at a given position. The signal CarO peptide sequence is included in this analysis. The conserved (C1-C4) regions, variable (VR1, VR2) and hypervariable (HVR1, HVR2) regions are shown. (B) Predicted transmembrane topology of the different <i>A</i>. <i>baumannii</i> CarO variants from CarO groups I, II and III. External loops (L1-L5), transmembrane spanning regions (β1-β10) and periplasmic regions are depicted.</p
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<p>Synthase genes present in genomes of <i>Br</i>. <i>laterosporus</i> BGSP7, BGSP9 and BGSP11 strains.</p
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<p><i>brvA</i>—brevibacillin synthetase A, <i>brvB</i>—brevibacillin synthetase B, <i>brvC</i>—brevibacillin synthetase C, <i>brvD</i>—brevibacillin synthetase D,... more
<p><i>brvA</i>—brevibacillin synthetase A, <i>brvB</i>—brevibacillin synthetase B, <i>brvC</i>—brevibacillin synthetase C, <i>brvD</i>—brevibacillin synthetase D, <i>brvE</i>—brevibacillin synthetase E, and <i>brvF</i>—brevibacillin ABC-transporter F genes.</p
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Antimicrobial activity of <i>Br</i>. <i>Laterosporus</i> BGSP7 (1), BGSP9 (2), BGSP11 (3) and BGSP12 (4) strains on different indicator strains: <i>S</i>. <i>aureus</i> ATCC25923 (A); <i>L</i>. <i>monocytogenes</i> ATCC19111 (B); <i>P</i>. <i>aeruginosa</i> MMA83 (C); <i>K</i>. <i>pneumoniae</i> ...more
<p>Antimicrobial activity was analysed after overnight growth (16 h) of the indicator strains.</p
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Functional characterization of the multidrug resistance CmbT transporter was performed in Lactococcus lactis. The cmbT gene is predicted to encode an efflux protein homologous to the multidrug resistance major facilitator superfamily. The... more
Functional characterization of the multidrug resistance CmbT transporter was performed in Lactococcus lactis. The cmbT gene is predicted to encode an efflux protein homologous to the multidrug resistance major facilitator superfamily. The cmbT gene (1377 bp) was cloned and overexpressed in L. lactis NZ9000. Results from cell growth studies revealed that the CmbT protein has an effect on host cell resistance to lincomycin, cholate, sulbactam, ethidium bromide, Hoechst 33342, sulfadiazine, streptomycin, rifampicin, puromycin and sulfametoxazole. Moreover, in vivo transport assays showed that overexpressed CmbT-mediated extrusion of ethidium bromide and Hoechst 33342 was higher than in the control L. lactis NZ9000 strain. CmbT-mediated extrusion of Hoechst 33342 was inhibited by the ionophores nigericin and valinomycin known to dissipate proton motive force. This indicates that CmbT-mediated extrusion is based on a drug-proton antiport mechanism. Taking together results obtained in thi...