144 research outputs found
LACTONASE MEDIATED QUORUM QUENCHING OF PSEUDOMONAS AERUGINOSA VIRULENCE
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 ā 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 formationand initiating the
decomposition of the preformed biofilm of P. aeruginosa MMA83.
Functional assays showed that lactonases have the ability to
significantly reduce extracellular virulence factors production ā
elastase, pyocyanin and rhamnolipid. Additionally, the results
obtained by real-time quantitative PCR showed that analyzed
recombinant enzymes significantly downregulated all three analyzed
P. aeruginosa QS networks at the transcriptional level. Finally, S.
maltophilia 6960 YtnP lactonase significantly prolonged survival of
Caenorhabditis elegans by reducing virulence of P. aeruginosa using fastkilling
liquid assay.
The described properties make B. cepacia and S. maltophilia lactonases
the promising therapeutic candidates for the development of nextgeneration
antivirulence agents.Book of abstracts and conference proceedings / 3rd International
Conference Antimicrobial Resistance - Current State and Perspectives, 16-18.
May 2024, Novi Sad, Serbia
Colistin resistance in acinetobacter baumannii: Molecular mechanisms and epidemiology
: Acinetobacter baumannii is recognized as a clinically significant pathogen causing a wide
spectrum of nosocomial infections. Colistin was considered a last-resort antibiotic for the treatment of
infections caused by multidrug-resistant A. baumannii. Since the reintroduction of colistin, a number
of mechanisms of colistin resistance in A. baumannii have been reported, including complete loss of
LPS by inactivation of the biosynthetic pathway, modifications of target LPS driven by the addition
of phosphoethanolamine (PEtN) moieties to lipid A mediated by the chromosomal pmrCAB operon
and eptA gene-encoded enzymes or plasmid-encoded mcr genes and efflux of colistin from the cell.
In addition to resistance to colistin, widespread heteroresistance is another feature of A. baumannii
that leads to colistin treatment failure. This review aims to present a critical assessment of relevant
published (>50 experimental papers) up-to-date knowledge on the molecular mechanisms of colistin
resistance in A. baumannii with a detailed review of implicated mutations and the global distribution
of colistin-resistant strains
Biogenic silencers of Pseudomonas aeruginosa virulence
Pseudomonas aeruginosa jedan je od najznaÄajnijih uzroÄnika unutarbolniÄkih infekcija Äiji je terapijski tretman
konvencionalnim antibioticima sve ÄeÅ”Äe neefikasan usled rezistencije na antibiotike. Inovativni vidovi
kontrole infekcija, poput utiÅ”avanja meÄuÄelijske komunikacije bakterija, a time i onemoguÄavanja virulencije
i inhibicije patogenog fenotipa su stoga od izuzetnog znaÄaja. U ovom radu biÄe predstavljena istraživanja
koja su bazirana na prirodnom svojstvu bakterija koje dele ekoloÅ”ke niÅ”e da saraÄuju, ali i kompetiraju,
na osnovu Äega 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 rekonstituiÅ”e osetljivost na antibiotike, a takoÄe je po prvi put u literaturi opisano prisustvo dihidroksi-
C18-HSL u bioloŔkim uzorcima. Opisane su i laktonaze vrste B. cepacia BCC4135 koje degraduju
autoinducere komunikacije P. aeruginosa i inhibiraju ekspresiju faktora virulencije. UtvrÄena je njihova supstratna
specifiÄnost i ukazano na razliÄitu bioloÅ”ku 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.Jedan deo ovog rada realizovan je na Dipartimento di Scienze Chimiche, UniversitĆ di Napoli Federico
II, Napulj, Italija pod rukovodstvom prof dr Antonio Molinaro i dr Flaviana Di Lorenzo, kojima se ovom prilikom
zahvaljujem. Hvala dr sci med Zorici VasiljeviÄ sa Instituta za zdravstvenu zaÅ”titu majke i deteta Srbije āāDr Vukan ÄupiÄāā
koja je obezbedila kliniÄke izolate koriÅ”Äene u ovom radu, kao i dr Milanu KojiÄu i drugim saradnicima Laboratorije za
molekularnu mikrobiologiju Instituta za molekularnu genetiku i genetiÄko inženjerstvo za izuzetnu pomoÄ tokom
izrade eksperimenata
Molecular characterization of semi-hard homemade cheese microflora
U poslednjoj deceniji zabeležen je nagli razvoj molekularnih tehnika baziranih na 16S i 23S rRNK, koje se koriste u izuÄavanju biodiverziteta mikroorganizama. U ovom radu ispitivana je mikroflora polutvrdog sira pripremljenog u domaÄinstvu. Zbog visokog sadržaja masti u ovom siru razvili smo novu tehniku za izolaciju totalne DNK iz sira (metod je baziran na bead beating-u). Brza izolacija DNK iz mikroflore sira omoguÄila nam je molekularnu identifikaciju BMK (BAKTERIJE MLEÄNE KISELINE) na osnovu umnožavanja gena za 16S rRNK PCR metodom. Za umnožavanje su koriÅ”Äeni prajmeri specifiÄni za gene 16S rRNK lakto-bacila, ali su uslovi PCR reakcije bili takvi da su omoguÄavali i umnožavanje gena 16S rRNK laktokoka. Rezultati RFLP analize pokazali su da mikrofloru sira pripremljenog u domaÄinstvu Äine predominantno laktokoke.This decade has shown an impressive development in the application of molecular techniques based on 16S and 23S rRNA genes to study the microbial diversity in various ecosystems. Microflora of semi-hard homemade cheese was examined in this work. We developed a novel technique for DNA extraction (a bead beating based method) due to high fat content of this cheese. Rapid extraction of DNA from cheese microflora enabled a molecular identification of the LAB (Lactic Acid Bacteria) strains based on PCR amplification of 16S RNA coding sequences. The specific primers for 76S RNA gene of lactobacilli were used for amplification. The PCR reaction was performed at lower temperature, where the specificity of the annealing reaction was reduced and lactococcal sequences of 16S RNA genes were also amplified. The results of RFLP analysis revealed that the microflora of Doboj homemade cheese encompases mostly lactococci
ACINETOBACTER BAUMANNII RESISTANT TO LAST-LINE ANTIBIOTICS: AN EMERGING THREAT IN THE WESTERN BALKANS
Acinetobacter baumannii is considered one of
the greatest threats to public health on a global
scale. This Gram-negative pathogen causes
severe infections, mostly of nosocomial origin,
with a high mortality rate. In recent years, the
rapid increase in the emergence and spread of
antibiotic resistance in A. baumannii has significantly
limited the effective therapeutic options
against infections caused by this bacterium.
The last-line antibiotics used in the treatment
of multidrug-resistant (MDR) A. baumannii
are carbapenems, tigecycline and polymyxins.
However, resistance to these antibiotics is
steadily increasing, especially to carbapenems,
leading to an extensively drug-resistant (XDR)
and even pandrug-resistant (PDR) phenotype
of A. baumannii. In 2021, the European Centre
for Disease Prevention and Control (ECDC) reported
that resistance of Acinetobacter spp. to
carbapenems reached 50% or more, mostly in
Southern and Eastern European countries. Although
the Western Balkans is a part of this region,
detailed studies on the epidemiology and
antibiotic resistance of A. baumannii are mainly
limited to Serbia and Croatia. In most cases, carbapenem
resistance in A. baumannii is due to
the production of carbapenemases, in particular
b-lactamases belonging to the class D known
as oxacillinases. The studies from the Western
Balkan countries revealed that besides the intrinsic
blaOXA-51-like gene, the most prevalent
acquired oxacillinase gene was the blaOXA-
24-like followed by the blaOXA-23-like, while
the blaOXA-58-like and metallo- b-lactamase
blaNDM-1 genes were less common. Although
significantly lower compared to carbapenem-resistant,
the number of A. baumannii isolates resistant
to tigecycline and colistin is on a continual
rise in the Western Balkans. As worldwide,
the main mechanism conferring tigecycline resistance
to A. baumannii from the Western Balkans
was overexpression of efflux pumps. Also,
the majority of reported alternations leading to
colistin resistance in A. baumannii were found in
the pmrCAB operon, which is responsible for the
modification of the colistin target, LPS.Book of abstract: From biotechnology to human and planetary health XIII congress of microbiologists of Serbia with international participation Mikromed regio 5, ums series 24: 4th ā 6th april 2024, Mona Plaza hotel, Belgrade, Serbi
Supplementary data for the article: VatiÄ, S.; MirkoviÄ, N.; MiloÅ”eviÄ, J. R.; JovÄiÄ, B.; PoloviÄ, N. Ä. Broad Range of Substrate Specificities in Papain and Fig Latex Enzymes Preparations Improve Enumeration of Listeria Monocytogenes. International Journal of Food Microbiology 2020, 334, 108851. https://doi.org/10.1016/j.ijfoodmicro.2020.108851
Supplementary material for: [https://doi.org/10.1016/j.ijfoodmicro.2020.108851]Related to published version: [https://cherry.chem.bg.ac.rs/handle/123456789/4090
Effect of methionine and cysteine deprivation on growth of different natural isolates of Lactobacillus spp. in chemically defined media
Cilj ovog rada je bio da se utvrdi sposobnost prirodnih izolata laktobacila,izolovanih iz razliÄitih ekoloÅ”kih niÅ”a da rastu u hemijski definisanom medijumu sa ili bez prisustva aminokiselina koje sadrže sumpor, metionin i/ili cistein. Dobijeni rezultati su pokazali da je esencijalna aminokiselina za rast izolata L. paracasei subsp. paracasei BGHN14 i BGSJ2-8 cistein, dok je za izolate BGHN40, BGCG31, BGHV54T, koji pripadaju vrsti L. plantarum-metionin. Metionin je esencijalna aminokiselina za rast soja L. rhamnosus BGHV58T. Ostali analizirani sojevi,kao Å”to su L. plantarum BGSJ3-18,BGZB19,BGHV52Ta i BGHV43T za svoj rast zahtevaju prisustvo obe aminokiseline u medijumu.The purpose of this study was to determine the ability of natural isolates of lactobacilli from different ecological niches to grow in a chemically defined medium in the presence or absence of sulphur-containing amino acids, methionine and/or cysteine. The obtained results indicate that cysteine is essential for growth of L. paracasei subsp. paracasei BGHN14 and BGSJ2-8, while methionine is essential for isolates BGHN40, BGCG31, and BGHV54T of the species L. plantarum. Methionine is also essential for growth of L. rhamnosus BGHV58T. Other analyzed strains, such as L. plantarum BGSJ3-18, BGZB19, BGHV52Ta, and BGHV43T, require the presence of both amino acids for their growth
Supplementary data for the article: MiloÅ”eviÄ, J.; PetriÄ, J.; JovÄiÄ, B.; JankoviÄ, B.; PoloviÄ, N. Exploring the Potential of Infrared Spectroscopy in Qualitative and Quantitative Monitoring of Ovalbumin Amyloid Fibrillation. Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy 2020, 229. https://doi.org/10.1016/j.saa.2019.117882
Supplementary material for: [https://doi.org/10.1016/j.saa.2019.117882]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/3884]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3895
Post-translational regulation of the RpoS and PsrA genes in pseudomonas putida WCS358: The role of ClpXP protease
RpoS i PsrA proteini su kljuÄni transkripcioni regulatori koji se kod pseudomonada aktiviraju u odgovoru na stacionarnu fazu rasta. Cilj ove studije bio je utvrÄivanje uloge ClpXP (ATP zavisna serin proteaza) u stabilnosti RpoS i PsrA proteina tokom razliÄitih faza rasta u Pseudomonas putida WCS358. "Western blot" analiza proteinskih ekstrakata P. putida WCS358 i P. putida WCS358 clpX::Km iz rane eksponencijalne, Kasne eksponencijalne i stacionarne faze rasta, sa antitelima na RpoS i PsrA, pokazala je da ClpXP degraduje RpoS i PsrA u ranoj eksponencijalnoj fazi rasta.The RpoS and PsrA proteins are key transcriptional regulators that are activated in response to the stationary phase of growth in pseudomonads. This study was designed to establish whether ClpXP (ATP-dependent serine protease) regulates levels of RpoS and PsrA in Pseudomonas putida WCS358. Western blot analysis of P. putida WCS358 protein extracts from the early exponentianl, late exponential, and stationary phases of growth with antibodies against RpoS and PsrA revealed that these proteins are degraded by ClpXP in the early exponential phase of growth. The obtained results demonstrate a role for ClpXP protease in post-translational regulation of proteins encoded by the rpoS and psrA genes in Pseudomonas spp
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