TY  - CONF
AU  - Ćurčić, Jovana
AU  - Jakovljević, Stefan
AU  - Novović, Katarina
AU  - Vasiljević, Zorica
AU  - Kojić, Milan
AU  - Jovčić, Branko
AU  - Malešević, Milka
PY  - 2023
UR  - http://intor.torlakinstitut.com/handle/123456789/803
AB  - Introduction: Quorum quenching (QQ) isthe enzymatic degradation of cell-to-cellsignaling molecules.
In this study, the potential of the novel YtnP lactonase, the quorum quenching enzyme derived from S.
maltophilia, to reduce P. aeruginosa quorum sensing and virulence factor gene expression was investigated.
Methods: MMA83 culture (adjusted to 1.5x105 CFU/ml) was treated with recombinant YtnP lactonase
(final concentration 50 μg/ml) at 37°C for 12 hours under aeration. RNA isolation of the treated and untreated MMA83 culture was performed using the RNeasy Mini Kit (Qiagen, Germany) according to the
protocol. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR), was used to analyze
the effect ofYtnP lactonase on the relative mRNA levels of the LasI/LasR, RhiI/RhiR, and PQS signaling network genes of P. aeruginosa MMA83 and virulence factor genes. The rpsL was used as an endogenous
control to normalize obtained data following the 2-ΔΔCt method.
Results: The QS genes belonging to three QS networks – LasI/LasR, RhiI/RhiR, and PQS of P. aeruginosa
MMA83 treated with YtnP lactonase were significantly downregulated. The RT -qPCR results show that
treatment with YtnP-lactonase decreased the relative mRNA levels of genes involved in the production
of elastase (lasB approximately 2-fold), alginate (algK approximately 2.2-fold), pyocyanin (phzM approximately 3.5-fold), pyoverdin (pvdS approximately 2-fold), and rhamnolipid (rhlC approximately 4-fold).
These results suggest that YtnP lactonase exerts an antivirulence effect at the transcription level.
Conclusion: YtnP lactonase, a quorum quenching (QQ) enzyme, has the potential to be used as an innovative enzyme-based antivirulence therapeutic to combat infections caused by P. aeruginosa.
PB  - Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade
C3  - CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia
T1  - A novel YtnP lactonase reduces the expression of p. aeruginosa MMA83 quorum sensing andvirulence factors gene expression
EP  - 121
SP  - 121
UR  - https://hdl.handle.net/21.15107/rcub_intor_803
ER  - 

@conference{
author = "Ćurčić, Jovana and Jakovljević, Stefan and Novović, Katarina and Vasiljević, Zorica and Kojić, Milan and Jovčić, Branko and Malešević, Milka",
year = "2023",
abstract = "Introduction: Quorum quenching (QQ) isthe enzymatic degradation of cell-to-cellsignaling molecules.
In this study, the potential of the novel YtnP lactonase, the quorum quenching enzyme derived from S.
maltophilia, to reduce P. aeruginosa quorum sensing and virulence factor gene expression was investigated.
Methods: MMA83 culture (adjusted to 1.5x105 CFU/ml) was treated with recombinant YtnP lactonase
(final concentration 50 μg/ml) at 37°C for 12 hours under aeration. RNA isolation of the treated and untreated MMA83 culture was performed using the RNeasy Mini Kit (Qiagen, Germany) according to the
protocol. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR), was used to analyze
the effect ofYtnP lactonase on the relative mRNA levels of the LasI/LasR, RhiI/RhiR, and PQS signaling network genes of P. aeruginosa MMA83 and virulence factor genes. The rpsL was used as an endogenous
control to normalize obtained data following the 2-ΔΔCt method.
Results: The QS genes belonging to three QS networks – LasI/LasR, RhiI/RhiR, and PQS of P. aeruginosa
MMA83 treated with YtnP lactonase were significantly downregulated. The RT -qPCR results show that
treatment with YtnP-lactonase decreased the relative mRNA levels of genes involved in the production
of elastase (lasB approximately 2-fold), alginate (algK approximately 2.2-fold), pyocyanin (phzM approximately 3.5-fold), pyoverdin (pvdS approximately 2-fold), and rhamnolipid (rhlC approximately 4-fold).
These results suggest that YtnP lactonase exerts an antivirulence effect at the transcription level.
Conclusion: YtnP lactonase, a quorum quenching (QQ) enzyme, has the potential to be used as an innovative enzyme-based antivirulence therapeutic to combat infections caused by P. aeruginosa.",
publisher = "Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade",
journal = "CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia",
title = "A novel YtnP lactonase reduces the expression of p. aeruginosa MMA83 quorum sensing andvirulence factors gene expression",
pages = "121-121",
url = "https://hdl.handle.net/21.15107/rcub_intor_803"
}

Ćurčić, J., Jakovljević, S., Novović, K., Vasiljević, Z., Kojić, M., Jovčić, B.,& Malešević, M.. (2023). A novel YtnP lactonase reduces the expression of p. aeruginosa MMA83 quorum sensing andvirulence factors gene expression. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia
Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade., 121-121.
https://hdl.handle.net/21.15107/rcub_intor_803

Ćurčić J, Jakovljević S, Novović K, Vasiljević Z, Kojić M, Jovčić B, Malešević M. A novel YtnP lactonase reduces the expression of p. aeruginosa MMA83 quorum sensing andvirulence factors gene expression. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia. 2023;:121-121.
https://hdl.handle.net/21.15107/rcub_intor_803 .

Ćurčić, Jovana, Jakovljević, Stefan, Novović, Katarina, Vasiljević, Zorica, Kojić, Milan, Jovčić, Branko, Malešević, Milka, "A novel YtnP lactonase reduces the expression of p. aeruginosa MMA83 quorum sensing andvirulence factors gene expression" in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia (2023):121-121,
https://hdl.handle.net/21.15107/rcub_intor_803 .

TY  - CONF
AU  - Gardijan, Lazar
AU  - Kojić, Milan
AU  - Jovanović, Goran
AU  - Malešević, Milka
PY  - 2023
UR  - http://intor.torlakinstitut.com/handle/123456789/804
AB  - Introduction: Lactolisterin BU (LBU) is a potent bacteriocin derived from Lactococcuslactis subsp. lactis
bv. diacetylactis BGBU1-4. It exhibits antimicrobial properties against Gram-positive food spoilage and
foodborne pathogens. This research aimed to explore the impact of amino acid substitution in LBU on
its antimicrobial activity by utilizing in silico prediction of LBU’ssecondary structure and amino acid substitutions.
Methods: The secondary structure of LBU was predicted using Phyre2 software. Five variants of LBU
were selected and chemically synthesized, along with unaltered LBU and BHT-B,serving as controls. Peptides were twofold diluted in distilled water, resulting in final concentrations ranging from 1000 µg/ml
to 0.5 µg/ml. An agarspot test, employing 5 µl of the dilution, was conducted on three indicatorstrains:
Lactococcus lactis BGMN1-596, Listeria monocytogenes ATCC19111, and Staphylococcus aureus
ATCC25923. The presence of inhibition zones was analyzed after overnight incubation at 37°C (S. aureus)
and 30°C (L. lactis and L. monocytogenes).
Results: Phyre2 analysis unveiled the presence of two α-helices in LBU’s structure. The majority of LBU
variants displayed altered antimicrobial activity, with some changes being genusspecific, potentially attributable to variances in cell wall composition. Some variants completely lost their activity, underscoring the significance of native amino acids or their physicochemical properties in the corresponding
positions within LBU’s structure. Furthermore, it was confirmed that chemically synthesized LBU effectively retains its antimicrobial activity.
Conclusion: Changesin amino acid composition give insight on structure-function relationship of LBU.
PB  - Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade
C3  - CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia
T1  - Influence of amino acid substitution on the antimicrobial activity of bacteriocin lactolisterin BU
EP  - 123
SP  - 123
UR  - https://hdl.handle.net/21.15107/rcub_intor_804
ER  - 

@conference{
author = "Gardijan, Lazar and Kojić, Milan and Jovanović, Goran and Malešević, Milka",
year = "2023",
abstract = "Introduction: Lactolisterin BU (LBU) is a potent bacteriocin derived from Lactococcuslactis subsp. lactis
bv. diacetylactis BGBU1-4. It exhibits antimicrobial properties against Gram-positive food spoilage and
foodborne pathogens. This research aimed to explore the impact of amino acid substitution in LBU on
its antimicrobial activity by utilizing in silico prediction of LBU’ssecondary structure and amino acid substitutions.
Methods: The secondary structure of LBU was predicted using Phyre2 software. Five variants of LBU
were selected and chemically synthesized, along with unaltered LBU and BHT-B,serving as controls. Peptides were twofold diluted in distilled water, resulting in final concentrations ranging from 1000 µg/ml
to 0.5 µg/ml. An agarspot test, employing 5 µl of the dilution, was conducted on three indicatorstrains:
Lactococcus lactis BGMN1-596, Listeria monocytogenes ATCC19111, and Staphylococcus aureus
ATCC25923. The presence of inhibition zones was analyzed after overnight incubation at 37°C (S. aureus)
and 30°C (L. lactis and L. monocytogenes).
Results: Phyre2 analysis unveiled the presence of two α-helices in LBU’s structure. The majority of LBU
variants displayed altered antimicrobial activity, with some changes being genusspecific, potentially attributable to variances in cell wall composition. Some variants completely lost their activity, underscoring the significance of native amino acids or their physicochemical properties in the corresponding
positions within LBU’s structure. Furthermore, it was confirmed that chemically synthesized LBU effectively retains its antimicrobial activity.
Conclusion: Changesin amino acid composition give insight on structure-function relationship of LBU.",
publisher = "Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade",
journal = "CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia",
title = "Influence of amino acid substitution on the antimicrobial activity of bacteriocin lactolisterin BU",
pages = "123-123",
url = "https://hdl.handle.net/21.15107/rcub_intor_804"
}

Gardijan, L., Kojić, M., Jovanović, G.,& Malešević, M.. (2023). Influence of amino acid substitution on the antimicrobial activity of bacteriocin lactolisterin BU. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia
Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade., 123-123.
https://hdl.handle.net/21.15107/rcub_intor_804

Gardijan L, Kojić M, Jovanović G, Malešević M. Influence of amino acid substitution on the antimicrobial activity of bacteriocin lactolisterin BU. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia. 2023;:123-123.
https://hdl.handle.net/21.15107/rcub_intor_804 .

Gardijan, Lazar, Kojić, Milan, Jovanović, Goran, Malešević, Milka, "Influence of amino acid substitution on the antimicrobial activity of bacteriocin lactolisterin BU" in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia (2023):123-123,
https://hdl.handle.net/21.15107/rcub_intor_804 .

TY  - JOUR
AU  - Filipić, Brankica
AU  - Malešević, Milka
AU  - Vasiljević, Zorica
AU  - Novović, Katarina
AU  - Kojić, Milan
AU  - Jovčić, Branko
PY  - 2022
UR  - http://intor.torlakinstitut.com/handle/123456789/724
AB  - 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.
PB  - Springer Science and Business Media B.V.
T2  - Folia Microbiologica
T1  - Comparative genomics of trimethoprim-sulfamethoxazole-resistant Achromobacter xylosoxidans clinical isolates from Serbia reveals shortened variant of class 1 integron integrase gene
DO  - 10.1007/s12223-022-01026-8
ER  - 

@article{
author = "Filipić, Brankica and Malešević, Milka and Vasiljević, Zorica and Novović, Katarina and Kojić, Milan and Jovčić, Branko",
year = "2022",
abstract = "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.",
publisher = "Springer Science and Business Media B.V.",
journal = "Folia Microbiologica",
title = "Comparative genomics of trimethoprim-sulfamethoxazole-resistant Achromobacter xylosoxidans clinical isolates from Serbia reveals shortened variant of class 1 integron integrase gene",
doi = "10.1007/s12223-022-01026-8"
}

Filipić, B., Malešević, M., Vasiljević, Z., Novović, K., Kojić, M.,& Jovčić, B.. (2022). Comparative genomics of trimethoprim-sulfamethoxazole-resistant Achromobacter xylosoxidans clinical isolates from Serbia reveals shortened variant of class 1 integron integrase gene. in Folia Microbiologica
Springer Science and Business Media B.V...
https://doi.org/10.1007/s12223-022-01026-8

Filipić B, Malešević M, Vasiljević Z, Novović K, Kojić M, Jovčić B. Comparative genomics of trimethoprim-sulfamethoxazole-resistant Achromobacter xylosoxidans clinical isolates from Serbia reveals shortened variant of class 1 integron integrase gene. in Folia Microbiologica. 2022;.
doi:10.1007/s12223-022-01026-8 .

Filipić, Brankica, Malešević, Milka, Vasiljević, Zorica, Novović, Katarina, Kojić, Milan, Jovčić, Branko, "Comparative genomics of trimethoprim-sulfamethoxazole-resistant Achromobacter xylosoxidans clinical isolates from Serbia reveals shortened variant of class 1 integron integrase gene" in Folia Microbiologica (2022),
https://doi.org/10.1007/s12223-022-01026-8 . .

TY  - JOUR
AU  - Vukotić, Goran
AU  - Obradović, Mina
AU  - Novović, Katarina
AU  - Di Luca, Mariagrazia
AU  - Jovčić, Branko
AU  - Fira, Đorđe
AU  - Neve, Horst
AU  - Kojić, Milan
AU  - McAuliffe, Olivia
PY  - 2020
UR  - http://intor.torlakinstitut.com/handle/123456789/721
AB  - Acinetobacter baumanniiis a leading cause of healthcare-associated infections worldwide. Its various intrinsic and acquired mechanisms of antibiotic resistance make the therapeutic challenge even more serious. One of the promising alternative treatments that is increasingly highlighted is phage therapy, the therapeutic use of bacteriophages to treat bacterial infections. Two phages active against nosocomial carbapenem-resistantA. baumanniistrain 6077/12, vB_AbaM_ISTD, and vB_AbaM_NOVI, were isolated from Belgrade wastewaters, purified, and concentrated using CsCl gradient ultracentrifugation. The phages were screened against 103 clinical isolates ofA. baumanniifrom a laboratory collection and characterized based on plaque and virion morphology, host range, adsorption rate, and one-step growth curve. Given that phage ISTD showed a broader host range, better adsorption rate, shorter latent period, and larger burst size, its ability to lyse planktonic and biofilm-embedded cells was tested in detail. Phage ISTD yielded a 3.5- and 2-log reduction in planktonic and biofilm-associated viable bacterial cell count, respectively, but the effect was time-dependent. Both phages produced growing turbid halos around plaques indicating the synthesis of depolymerases, enzymes capable of degrading bacterial exopolysaccharides. Halos tested positive for presence of phages in the proximity of the plaque, but not further from the plaque, which indicates that the observed halo enlargement is a consequence of enzyme diffusion through the agar, independently of the phages. This notion was also supported by the growing halos induced by phage preparations applied on pregrown bacterial lawns, indicating that depolymerizing effect was achieved also on non-dividing sensitive cells. Overall, good rates of growth, fast adsorption rate, broad host range, and high depolymerizing activity, as well as antibacterial effectiveness against planktonic and biofilm-associated bacteria, make these phages good candidates for potential application in combatingA. baumanniiinfections.
PB  - Frontiers Media Sa, Lausanne
T2  - Frontiers in Medicine
T1  - Characterization, Antibiofilm, and Depolymerizing Activity of Two Phages Active on Carbapenem-ResistantAcinetobacter baumannii
VL  - 7
DO  - 10.3389/fmed.2020.00426
ER  - 

@article{
author = "Vukotić, Goran and Obradović, Mina and Novović, Katarina and Di Luca, Mariagrazia and Jovčić, Branko and Fira, Đorđe and Neve, Horst and Kojić, Milan and McAuliffe, Olivia",
year = "2020",
abstract = "Acinetobacter baumanniiis a leading cause of healthcare-associated infections worldwide. Its various intrinsic and acquired mechanisms of antibiotic resistance make the therapeutic challenge even more serious. One of the promising alternative treatments that is increasingly highlighted is phage therapy, the therapeutic use of bacteriophages to treat bacterial infections. Two phages active against nosocomial carbapenem-resistantA. baumanniistrain 6077/12, vB_AbaM_ISTD, and vB_AbaM_NOVI, were isolated from Belgrade wastewaters, purified, and concentrated using CsCl gradient ultracentrifugation. The phages were screened against 103 clinical isolates ofA. baumanniifrom a laboratory collection and characterized based on plaque and virion morphology, host range, adsorption rate, and one-step growth curve. Given that phage ISTD showed a broader host range, better adsorption rate, shorter latent period, and larger burst size, its ability to lyse planktonic and biofilm-embedded cells was tested in detail. Phage ISTD yielded a 3.5- and 2-log reduction in planktonic and biofilm-associated viable bacterial cell count, respectively, but the effect was time-dependent. Both phages produced growing turbid halos around plaques indicating the synthesis of depolymerases, enzymes capable of degrading bacterial exopolysaccharides. Halos tested positive for presence of phages in the proximity of the plaque, but not further from the plaque, which indicates that the observed halo enlargement is a consequence of enzyme diffusion through the agar, independently of the phages. This notion was also supported by the growing halos induced by phage preparations applied on pregrown bacterial lawns, indicating that depolymerizing effect was achieved also on non-dividing sensitive cells. Overall, good rates of growth, fast adsorption rate, broad host range, and high depolymerizing activity, as well as antibacterial effectiveness against planktonic and biofilm-associated bacteria, make these phages good candidates for potential application in combatingA. baumanniiinfections.",
publisher = "Frontiers Media Sa, Lausanne",
journal = "Frontiers in Medicine",
title = "Characterization, Antibiofilm, and Depolymerizing Activity of Two Phages Active on Carbapenem-ResistantAcinetobacter baumannii",
volume = "7",
doi = "10.3389/fmed.2020.00426"
}

Vukotić, G., Obradović, M., Novović, K., Di Luca, M., Jovčić, B., Fira, Đ., Neve, H., Kojić, M.,& McAuliffe, O.. (2020). Characterization, Antibiofilm, and Depolymerizing Activity of Two Phages Active on Carbapenem-ResistantAcinetobacter baumannii. in Frontiers in Medicine
Frontiers Media Sa, Lausanne., 7.
https://doi.org/10.3389/fmed.2020.00426

Vukotić G, Obradović M, Novović K, Di Luca M, Jovčić B, Fira Đ, Neve H, Kojić M, McAuliffe O. Characterization, Antibiofilm, and Depolymerizing Activity of Two Phages Active on Carbapenem-ResistantAcinetobacter baumannii. in Frontiers in Medicine. 2020;7.
doi:10.3389/fmed.2020.00426 .

Vukotić, Goran, Obradović, Mina, Novović, Katarina, Di Luca, Mariagrazia, Jovčić, Branko, Fira, Đorđe, Neve, Horst, Kojić, Milan, McAuliffe, Olivia, "Characterization, Antibiofilm, and Depolymerizing Activity of Two Phages Active on Carbapenem-ResistantAcinetobacter baumannii" in Frontiers in Medicine, 7 (2020),
https://doi.org/10.3389/fmed.2020.00426 . .