TY  - CONF
AU  - Šapić, Katarina
AU  - Novović, Katarina
AU  - Radovanović, Milica
AU  - Gajić, Ina
AU  - Vasiljević, Zorica
AU  - Malešević, Milka
AU  - Jovčić, Branko
PY  - 2024
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/2386
AB  - 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 enables tigecycline resistance in clinical
A. baumannii isolates.
PB  - Serbian Society for Microbiology
C3  - XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health
T1  - THE ROLE OF EFFLUX PUMPS IN TIGECYCLINE RESISTANCE OF ACINETOBACTER BAUMANNII ISOLATES FROM WESTERN BALKAN HOSPITALS
EP  - 187
SP  - 187
UR  - https://hdl.handle.net/21.15107/rcub_imagine_2386
ER  - 

@conference{
author = "Šapić, Katarina and Novović, Katarina and Radovanović, Milica and Gajić, Ina and Vasiljević, Zorica and Malešević, Milka and Jovčić, Branko",
year = "2024",
abstract = "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 enables tigecycline resistance in clinical
A. baumannii isolates.",
publisher = "Serbian Society for Microbiology",
journal = "XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health",
title = "THE ROLE OF EFFLUX PUMPS IN TIGECYCLINE RESISTANCE OF ACINETOBACTER BAUMANNII ISOLATES FROM WESTERN BALKAN HOSPITALS",
pages = "187-187",
url = "https://hdl.handle.net/21.15107/rcub_imagine_2386"
}

Šapić, K., Novović, K., Radovanović, M., Gajić, I., Vasiljević, Z., Malešević, M.,& Jovčić, B.. (2024). THE ROLE OF EFFLUX PUMPS IN TIGECYCLINE RESISTANCE OF ACINETOBACTER BAUMANNII ISOLATES FROM WESTERN BALKAN HOSPITALS. in XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health
Serbian Society for Microbiology., 187-187.
https://hdl.handle.net/21.15107/rcub_imagine_2386

Šapić K, Novović K, Radovanović M, Gajić I, Vasiljević Z, Malešević M, Jovčić B. THE ROLE OF EFFLUX PUMPS IN TIGECYCLINE RESISTANCE OF ACINETOBACTER BAUMANNII ISOLATES FROM WESTERN BALKAN HOSPITALS. in XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health. 2024;:187-187.
https://hdl.handle.net/21.15107/rcub_imagine_2386 .

Šapić, Katarina, Novović, Katarina, Radovanović, Milica, Gajić, Ina, Vasiljević, Zorica, Malešević, Milka, Jovčić, Branko, "THE ROLE OF EFFLUX PUMPS IN TIGECYCLINE RESISTANCE OF ACINETOBACTER BAUMANNII ISOLATES FROM WESTERN BALKAN HOSPITALS" in XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health (2024):187-187,
https://hdl.handle.net/21.15107/rcub_imagine_2386 .

TY  - CONF
AU  - Plačkić, Nikola
AU  - Kljajević, Nemanja
AU  - Obradović, Mina
AU  - Kekić, Dušan
AU  - Gajić, Ina
AU  - Stanisavljević, Nemanja
AU  - Vukotić, Goran
PY  - 2024
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/2383
AB  - Anti-microbial drug resistance (AMR) is one of
the global health threats caused by the misuse
of drugs typically used to treat microbial
infections in humans, animals and plants. AMR
in nosocomial infections not only significantly
hinders treatment and endangers the patients’
lives, but also elevates the costs of healthcare.
Multiple research approaches have been initiated
to combat AMR, and one promising method
is bacteriophage therapy. Bacteriophages (phages)
are viruses that naturally exploit bacteria as
their hosts for replication and can cause cell lysis,
which makes them promising candidates for
treating the infections that do not respond to
conventional antibiotic therapies. In this study,
we screened wastewater samples from four
different collectors in Belgrade urban area for
bacteriophages active against clinically isolated
strains of two biofilm-producing bacteria that
readily persist in hospital environment - Klebsiella
pneumoniae (6 strains) and Pseudomonas aeruginosa
(2 strains). Wastewaters were screened
for phage presence through phage enrichment
process, in which bacteria were grown in a mixture
of water samples and nutrient-rich broth.
Obtained cultures were screened for antimicrobial
activity against the respective host strains,
and candidates were subjected to a first-round
plaque assay to detect the phages. Finally, the
activity of all the candidates was tested against
all strains of the same species to gain the first insight
into their host range. We discovered 20 potentially
distinct bacteriophages active against
K. pneumoniae strains and two potentially different
candidates targeting P. aeruginosa. Notably,
one phage exhibited activity against all tested K.
pneumoniae strains, and four were active against
5 out of 6 tested strains. Among 22 candidates in
total, five showed depolymerizing activity, indicating
promise in combating biofilm formation.
Currently, isolation of new phages, as well as purification
and host range analysis is underway for
several candidates targeting K. pneumoniae and
two targeting P. aeruginosa strains.
PB  - Serbian Society for Microbiology
C3  - XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health
T1  - NOVEL BACTERIOPHAGE ISOLATION FROM BELGRADE WASTEWATERS
EP  - 148
SP  - 148
UR  - https://hdl.handle.net/21.15107/rcub_imagine_2383
ER  - 

@conference{
author = "Plačkić, Nikola and Kljajević, Nemanja and Obradović, Mina and Kekić, Dušan and Gajić, Ina and Stanisavljević, Nemanja and Vukotić, Goran",
year = "2024",
abstract = "Anti-microbial drug resistance (AMR) is one of
the global health threats caused by the misuse
of drugs typically used to treat microbial
infections in humans, animals and plants. AMR
in nosocomial infections not only significantly
hinders treatment and endangers the patients’
lives, but also elevates the costs of healthcare.
Multiple research approaches have been initiated
to combat AMR, and one promising method
is bacteriophage therapy. Bacteriophages (phages)
are viruses that naturally exploit bacteria as
their hosts for replication and can cause cell lysis,
which makes them promising candidates for
treating the infections that do not respond to
conventional antibiotic therapies. In this study,
we screened wastewater samples from four
different collectors in Belgrade urban area for
bacteriophages active against clinically isolated
strains of two biofilm-producing bacteria that
readily persist in hospital environment - Klebsiella
pneumoniae (6 strains) and Pseudomonas aeruginosa
(2 strains). Wastewaters were screened
for phage presence through phage enrichment
process, in which bacteria were grown in a mixture
of water samples and nutrient-rich broth.
Obtained cultures were screened for antimicrobial
activity against the respective host strains,
and candidates were subjected to a first-round
plaque assay to detect the phages. Finally, the
activity of all the candidates was tested against
all strains of the same species to gain the first insight
into their host range. We discovered 20 potentially
distinct bacteriophages active against
K. pneumoniae strains and two potentially different
candidates targeting P. aeruginosa. Notably,
one phage exhibited activity against all tested K.
pneumoniae strains, and four were active against
5 out of 6 tested strains. Among 22 candidates in
total, five showed depolymerizing activity, indicating
promise in combating biofilm formation.
Currently, isolation of new phages, as well as purification
and host range analysis is underway for
several candidates targeting K. pneumoniae and
two targeting P. aeruginosa strains.",
publisher = "Serbian Society for Microbiology",
journal = "XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health",
title = "NOVEL BACTERIOPHAGE ISOLATION FROM BELGRADE WASTEWATERS",
pages = "148-148",
url = "https://hdl.handle.net/21.15107/rcub_imagine_2383"
}

Plačkić, N., Kljajević, N., Obradović, M., Kekić, D., Gajić, I., Stanisavljević, N.,& Vukotić, G.. (2024). NOVEL BACTERIOPHAGE ISOLATION FROM BELGRADE WASTEWATERS. in XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health
Serbian Society for Microbiology., 148-148.
https://hdl.handle.net/21.15107/rcub_imagine_2383

Plačkić N, Kljajević N, Obradović M, Kekić D, Gajić I, Stanisavljević N, Vukotić G. NOVEL BACTERIOPHAGE ISOLATION FROM BELGRADE WASTEWATERS. in XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health. 2024;:148-148.
https://hdl.handle.net/21.15107/rcub_imagine_2383 .

Plačkić, Nikola, Kljajević, Nemanja, Obradović, Mina, Kekić, Dušan, Gajić, Ina, Stanisavljević, Nemanja, Vukotić, Goran, "NOVEL BACTERIOPHAGE ISOLATION FROM BELGRADE WASTEWATERS" in XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health (2024):148-148,
https://hdl.handle.net/21.15107/rcub_imagine_2383 .

TY  - CONF
AU  - Vukotić, Goran
AU  - Obradović, Mina
AU  - Plačkić, Nikola
AU  - Kljajević, Nemanja
AU  - Pavić, Aleksandar
AU  - Kekić, Dušan
AU  - Gajić, Ina
AU  - Kojić, Milan
AU  - Stanisavljević, Nemanja
PY  - 2024
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/2375
AB  - Antimicrobial resistance (AMR) arises when
bacteria and other microbes stop responding
to medications. AMR is now recognized as one
of serious global health threats, repeatedly
appearing in the World Health Organization’s
(WHO) lists of urgent global health challenges,
including the 2024 list. It is taking a fatal toll
– nearly 5 million deaths globally per year are
associated with AMR, encompassing 1.27 million
directly attributed to AMR. The COVID-19
pandemic paved the way for aggravation of
bacterial AMR – primarily due to enhancement
in unspecific and unjustified prescription and
use of broad-spectrum antibiotics, resulting in
what is now recognized as „silent pandemic of
AMR“. Bacteriophages (phages) are natural and
specific predators of bacteria - viruses that can
infect, replicate inside and lyse arguably any
bacteria. Their therapeutic potential is being
hastily evaluated through different approaches:
in silico, in vitro, ex vivo and in vivo – in laboratory
animals as well as in human case and clinical
studies. Although the results are promising,bacteria rapidly develop resistance against
phages, which why the isolation and research
of new phages is needed. Our work is concentrated
on three bacterial species for which critical
priority by WHO has been declared – carbapenem-
resistant Acinetobacter baumannii,
Pseudomonas aeruginosa and Klebsiella pneumoniae.
Twenty distinct pathogenic strains of
A. baumannii, 6 K. pneumoniae and 6 P. aeruginosa
were used as targets for bacteriophage
isolation, and total of 14, 22 and 8 potentially
distinct phages were collected, respectively. All
strains were nosocomial isolates obtained from
various tissues, including from terminally ill patients.
Six phages were characterized in detail.
In particular, phage vB_AbaM_ISTD was applied
against A. baumannii in zebrafish embryo
model of systemic infection, and demonstrated
powerful therapeutic potential, eradicating the
infection. Interestingly, its DNA was characterized
with highly modified thymidine (amassing
1228 Da), making it the largest non-canonical
deoxynucleoside reported so far.
PB  - Serbian Society for Microbiology
C3  - XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health
T1  - BACTERIOPHAGES OF MULTIDRUG-RESISTANT NOSOCOMIAL PATHOGENS – BELGRADE EXPERIENCE
EP  - 121
SP  - 121
UR  - https://hdl.handle.net/21.15107/rcub_imagine_2375
ER  - 

@conference{
author = "Vukotić, Goran and Obradović, Mina and Plačkić, Nikola and Kljajević, Nemanja and Pavić, Aleksandar and Kekić, Dušan and Gajić, Ina and Kojić, Milan and Stanisavljević, Nemanja",
year = "2024",
abstract = "Antimicrobial resistance (AMR) arises when
bacteria and other microbes stop responding
to medications. AMR is now recognized as one
of serious global health threats, repeatedly
appearing in the World Health Organization’s
(WHO) lists of urgent global health challenges,
including the 2024 list. It is taking a fatal toll
– nearly 5 million deaths globally per year are
associated with AMR, encompassing 1.27 million
directly attributed to AMR. The COVID-19
pandemic paved the way for aggravation of
bacterial AMR – primarily due to enhancement
in unspecific and unjustified prescription and
use of broad-spectrum antibiotics, resulting in
what is now recognized as „silent pandemic of
AMR“. Bacteriophages (phages) are natural and
specific predators of bacteria - viruses that can
infect, replicate inside and lyse arguably any
bacteria. Their therapeutic potential is being
hastily evaluated through different approaches:
in silico, in vitro, ex vivo and in vivo – in laboratory
animals as well as in human case and clinical
studies. Although the results are promising,bacteria rapidly develop resistance against
phages, which why the isolation and research
of new phages is needed. Our work is concentrated
on three bacterial species for which critical
priority by WHO has been declared – carbapenem-
resistant Acinetobacter baumannii,
Pseudomonas aeruginosa and Klebsiella pneumoniae.
Twenty distinct pathogenic strains of
A. baumannii, 6 K. pneumoniae and 6 P. aeruginosa
were used as targets for bacteriophage
isolation, and total of 14, 22 and 8 potentially
distinct phages were collected, respectively. All
strains were nosocomial isolates obtained from
various tissues, including from terminally ill patients.
Six phages were characterized in detail.
In particular, phage vB_AbaM_ISTD was applied
against A. baumannii in zebrafish embryo
model of systemic infection, and demonstrated
powerful therapeutic potential, eradicating the
infection. Interestingly, its DNA was characterized
with highly modified thymidine (amassing
1228 Da), making it the largest non-canonical
deoxynucleoside reported so far.",
publisher = "Serbian Society for Microbiology",
journal = "XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health",
title = "BACTERIOPHAGES OF MULTIDRUG-RESISTANT NOSOCOMIAL PATHOGENS – BELGRADE EXPERIENCE",
pages = "121-121",
url = "https://hdl.handle.net/21.15107/rcub_imagine_2375"
}

Vukotić, G., Obradović, M., Plačkić, N., Kljajević, N., Pavić, A., Kekić, D., Gajić, I., Kojić, M.,& Stanisavljević, N.. (2024). BACTERIOPHAGES OF MULTIDRUG-RESISTANT NOSOCOMIAL PATHOGENS – BELGRADE EXPERIENCE. in XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health
Serbian Society for Microbiology., 121-121.
https://hdl.handle.net/21.15107/rcub_imagine_2375

Vukotić G, Obradović M, Plačkić N, Kljajević N, Pavić A, Kekić D, Gajić I, Kojić M, Stanisavljević N. BACTERIOPHAGES OF MULTIDRUG-RESISTANT NOSOCOMIAL PATHOGENS – BELGRADE EXPERIENCE. in XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health. 2024;:121-121.
https://hdl.handle.net/21.15107/rcub_imagine_2375 .

Vukotić, Goran, Obradović, Mina, Plačkić, Nikola, Kljajević, Nemanja, Pavić, Aleksandar, Kekić, Dušan, Gajić, Ina, Kojić, Milan, Stanisavljević, Nemanja, "BACTERIOPHAGES OF MULTIDRUG-RESISTANT NOSOCOMIAL PATHOGENS – BELGRADE EXPERIENCE" in XIII Congress of microbiologists of Serbia: From biotechnology to human and planetary health (2024):121-121,
https://hdl.handle.net/21.15107/rcub_imagine_2375 .