TY  - CONF
AU  - Novović, Katarina
AU  - Radovanović, Milica
AU  - Milić, Vukašin
AU  - Gajić, Ina
AU  - Vasiljević, Zorica
AU  - Jovčić, Branko
PY  - 2023
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/2134
AB  - Introduction: Multidrug-resistant (MDR) Acinetobacter baumannii has been recognized as one of the
most serious healthcare challenges worldwide. Although tigecycline represents one of the last resort
therapies for MDR A. baumannii, resistance to this antibiotic has been reported and mostly is mediated
by AdeABC efflux pump. The aim of our study was to investigate the molecular mechanism responsible
for tigecycline resistance of thirty-seven A. baumannii isolates from Balkan medical settings (Serbia,
Bosnia and Herzegovina and Montenegro) gathered in 2016 and 2022.
Methods: Minimal inhibitory concentration (MIC) values for tigecycline were determined using microdilution method according to EUCAST guidelines. Inhibition of the efflux of tigecycline wastested by
the same method using a combination of antibiotic and efflux pump inhibitor (CCCP). Amino acid alternations within AdeS and AdeR proteins were detected by comparing to sequences of referent isolates
ATCC19606 and ATCC17978. Expression of the adeB gene ofselected isolates was monitored by RT-qPCR.
Results: All tested isolates were resistant to tigecycline and showed significant decrease in tigecycline
MIC values in presence of CCCP (≥16-fold reduction) indicating that antibiotic efflux is responsible for
tigecycline resistance. The analysis of two-component system AdeRS, regulatory system of RND efflux
pump AdeABC, 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). In addition, RT-qPCR showed that
selected isolates upregulate expression of the adeB gene (from 1,13- to 3-fold).
Conclusion: This study revealed that AdeABC overexpression is the main mechanism of tigecycline resistance in A. baumannii isolated in Balkan hospitals.
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  - AdeABC efflux pump-mediated resistance to tigecycline in Acinetobacter baumannii isolates from Balkan hospitals
EP  - 154
SP  - 154
UR  - https://hdl.handle.net/21.15107/rcub_imagine_2134
ER  - 

@conference{
author = "Novović, Katarina and Radovanović, Milica and Milić, Vukašin and Gajić, Ina and Vasiljević, Zorica and Jovčić, Branko",
year = "2023",
abstract = "Introduction: Multidrug-resistant (MDR) Acinetobacter baumannii has been recognized as one of the
most serious healthcare challenges worldwide. Although tigecycline represents one of the last resort
therapies for MDR A. baumannii, resistance to this antibiotic has been reported and mostly is mediated
by AdeABC efflux pump. The aim of our study was to investigate the molecular mechanism responsible
for tigecycline resistance of thirty-seven A. baumannii isolates from Balkan medical settings (Serbia,
Bosnia and Herzegovina and Montenegro) gathered in 2016 and 2022.
Methods: Minimal inhibitory concentration (MIC) values for tigecycline were determined using microdilution method according to EUCAST guidelines. Inhibition of the efflux of tigecycline wastested by
the same method using a combination of antibiotic and efflux pump inhibitor (CCCP). Amino acid alternations within AdeS and AdeR proteins were detected by comparing to sequences of referent isolates
ATCC19606 and ATCC17978. Expression of the adeB gene ofselected isolates was monitored by RT-qPCR.
Results: All tested isolates were resistant to tigecycline and showed significant decrease in tigecycline
MIC values in presence of CCCP (≥16-fold reduction) indicating that antibiotic efflux is responsible for
tigecycline resistance. The analysis of two-component system AdeRS, regulatory system of RND efflux
pump AdeABC, 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). In addition, RT-qPCR showed that
selected isolates upregulate expression of the adeB gene (from 1,13- to 3-fold).
Conclusion: This study revealed that AdeABC overexpression is the main mechanism of tigecycline resistance in A. baumannii isolated in Balkan hospitals.",
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 = "AdeABC efflux pump-mediated resistance to tigecycline in Acinetobacter baumannii isolates from Balkan hospitals",
pages = "154-154",
url = "https://hdl.handle.net/21.15107/rcub_imagine_2134"
}

Novović, K., Radovanović, M., Milić, V., Gajić, I., Vasiljević, Z.,& Jovčić, B.. (2023). AdeABC efflux pump-mediated resistance to tigecycline in Acinetobacter baumannii isolates from Balkan hospitals. 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., 154-154.
https://hdl.handle.net/21.15107/rcub_imagine_2134

Novović K, Radovanović M, Milić V, Gajić I, Vasiljević Z, Jovčić B. AdeABC efflux pump-mediated resistance to tigecycline in Acinetobacter baumannii isolates from Balkan hospitals. 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;:154-154.
https://hdl.handle.net/21.15107/rcub_imagine_2134 .

Novović, Katarina, Radovanović, Milica, Milić, Vukašin, Gajić, Ina, Vasiljević, Zorica, Jovčić, Branko, "AdeABC efflux pump-mediated resistance to tigecycline in Acinetobacter baumannii isolates from Balkan hospitals" 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):154-154,
https://hdl.handle.net/21.15107/rcub_imagine_2134 .

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
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/2124
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 theprotocol. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR), was used to analyzethe 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 endogenouscontrol 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. aeruginosaMMA83 treated with YtnP lactonase were significantly downregulated. The RT -qPCR results show thattreatment with YtnP-lactonase decreased the relative mRNA levels of genes involved in the productionof 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_imagine_2124
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 theprotocol. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR), was used to analyzethe 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 endogenouscontrol 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. aeruginosaMMA83 treated with YtnP lactonase were significantly downregulated. The RT -qPCR results show thattreatment with YtnP-lactonase decreased the relative mRNA levels of genes involved in the productionof 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_imagine_2124"
}

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_imagine_2124

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_imagine_2124 .

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_imagine_2124 .

TY  - JOUR
AU  - Malešević, Milka
AU  - Stanisavljević, Nemanja
AU  - Novović, Katarina
AU  - Polović, Natalija
AU  - Vasiljević, Zorica
AU  - Kojić, Milan
AU  - Jovčić, Branko
PY  - 2020
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/1338
AB  - Burkholderia cepacia is well known as the causative agent of infections in humans where often shares niche with other pathogens, like Pseudomonas aeruginosa. Clinical isolate Burkholderia sp. BCC4135 was selected due to its strong quorum quenching (QQ) activity. Whole genome sequencing unveiled this isolate as B. cepacia with unique sequence type ST1485 and a myriad of genes belonging to resistome and virulome. Two QQ lactonases YtnP and Y2-aiiA originated from B. cepacia BCC4135 were cloned, expressed, and functionally characterized. They were active against a broad substrate spectrum of the N-acyl-homoserine lactones (AHLs). The YtnP lactonase was inactive, while Y2-aiiA was active against N-tetradecanoyl-DL-homoserine lactone (C14-HSL) which could imply the difference in their biological roles from the aspect of its quorum sensing (QS) autoregulation and interference with the QS systems of bacteria residing within the same niche. Both YtnP and Y2-aiiA were able to attenuate virulence potential of P. aeruginosa MMA83 clinical isolate declining its biofilm formation and virulence factors production. B. cepacia BCC4135 lactonases interfered with the las, rhl, and even pqs QS circuit of P. aeruginosa MMA83 transcription and the effect of combined enzymes was even more prominent. B. cepacia BCC4135 also employs the CepI/R QS system for governing its own virulence traits and possibly self-regulates the QQ/QS network through the different expression and activity of YtnP and/or Y2-aiiA. Our findings pointed out that BCC4135 lactonases could be exploited as an effective antivirulence drugs against P. aeruginosa and gave us a new insight into B. cepacia QQ/QS machinery.
PB  - Academic Press Ltd- Elsevier Science Ltd, London
T2  - Microbial Pathogenesis
T1  - Burkholderia cepacia YtnP and Y2-aiiA lactonases inhibit virulence of Pseudomonas aeruginosa via quorum quenching activity
VL  - 149
DO  - 10.1016/j.micpath.2020.104561
ER  - 

@article{
author = "Malešević, Milka and Stanisavljević, Nemanja and Novović, Katarina and Polović, Natalija and Vasiljević, Zorica and Kojić, Milan and Jovčić, Branko",
year = "2020",
abstract = "Burkholderia cepacia is well known as the causative agent of infections in humans where often shares niche with other pathogens, like Pseudomonas aeruginosa. Clinical isolate Burkholderia sp. BCC4135 was selected due to its strong quorum quenching (QQ) activity. Whole genome sequencing unveiled this isolate as B. cepacia with unique sequence type ST1485 and a myriad of genes belonging to resistome and virulome. Two QQ lactonases YtnP and Y2-aiiA originated from B. cepacia BCC4135 were cloned, expressed, and functionally characterized. They were active against a broad substrate spectrum of the N-acyl-homoserine lactones (AHLs). The YtnP lactonase was inactive, while Y2-aiiA was active against N-tetradecanoyl-DL-homoserine lactone (C14-HSL) which could imply the difference in their biological roles from the aspect of its quorum sensing (QS) autoregulation and interference with the QS systems of bacteria residing within the same niche. Both YtnP and Y2-aiiA were able to attenuate virulence potential of P. aeruginosa MMA83 clinical isolate declining its biofilm formation and virulence factors production. B. cepacia BCC4135 lactonases interfered with the las, rhl, and even pqs QS circuit of P. aeruginosa MMA83 transcription and the effect of combined enzymes was even more prominent. B. cepacia BCC4135 also employs the CepI/R QS system for governing its own virulence traits and possibly self-regulates the QQ/QS network through the different expression and activity of YtnP and/or Y2-aiiA. Our findings pointed out that BCC4135 lactonases could be exploited as an effective antivirulence drugs against P. aeruginosa and gave us a new insight into B. cepacia QQ/QS machinery.",
publisher = "Academic Press Ltd- Elsevier Science Ltd, London",
journal = "Microbial Pathogenesis",
title = "Burkholderia cepacia YtnP and Y2-aiiA lactonases inhibit virulence of Pseudomonas aeruginosa via quorum quenching activity",
volume = "149",
doi = "10.1016/j.micpath.2020.104561"
}

Malešević, M., Stanisavljević, N., Novović, K., Polović, N., Vasiljević, Z., Kojić, M.,& Jovčić, B.. (2020). Burkholderia cepacia YtnP and Y2-aiiA lactonases inhibit virulence of Pseudomonas aeruginosa via quorum quenching activity. in Microbial Pathogenesis
Academic Press Ltd- Elsevier Science Ltd, London., 149.
https://doi.org/10.1016/j.micpath.2020.104561

Malešević M, Stanisavljević N, Novović K, Polović N, Vasiljević Z, Kojić M, Jovčić B. Burkholderia cepacia YtnP and Y2-aiiA lactonases inhibit virulence of Pseudomonas aeruginosa via quorum quenching activity. in Microbial Pathogenesis. 2020;149.
doi:10.1016/j.micpath.2020.104561 .

Malešević, Milka, Stanisavljević, Nemanja, Novović, Katarina, Polović, Natalija, Vasiljević, Zorica, Kojić, Milan, Jovčić, Branko, "Burkholderia cepacia YtnP and Y2-aiiA lactonases inhibit virulence of Pseudomonas aeruginosa via quorum quenching activity" in Microbial Pathogenesis, 149 (2020),
https://doi.org/10.1016/j.micpath.2020.104561 . .