50 research outputs found
Identification and characterization of biogenic silencers of Pseudomonas aeruginosa quorum sensing
NemoguÄnost kontrole pojave i Å”irenja rezistencije na antibiotike usmerila je istraživanja u
poslednje dve dekade ka iznalaženju novih terapeutskih opcija, sa ciljem tretmana infekcija izazvanih
patogenim bakterijama rezistentnim na veÄi broj kliniÄki znaÄajnih antibiotika. UzevÅ”i u obzir
Äinjenicu da je antibiotska rezistencija kompleksan, multifaktorijalni fenomen, reÅ”enje ovog problema
ukljuÄuje niz pristupa usmerenih na kontrolu faktora koji olakÅ”avaju nastanak i Å”irenje rezistencije.
Jedan od tih pristupa sastoji se u razvoju novih terapeutika koji bi delovali mehanizmima razliÄitim od
trenutno dostupnih antibiotika. U tom pogledu, antivirulentna terapija zamiÅ”ljena je kao obeÄavajuÄa
alternativa sa ciljem kontrole virulencije specifiÄne za odreÄene patogene, bez vrÅ”enja snažnog
selektivnog pritiska na bakterijske Äelije. ImajuÄi u vidu da je Pseudomonas aeruginosa jedan od
vodeÄih uzroÄnika unutarbolniÄkih infekcija Å”irom sveta, leÄenje infekcija izazvanih ovim patogenom
predstavlja veliki terapeutski izazov. P. aeruginosa svoj patogeni potencijal ostvaruje zahvaljujuÄi
mnogobrojnim uroÄenim, steÄenim i adaptivnim mehanizmima rezistencije. Pored toga, posedovanje
sistema meÄuÄelijske komunikacije (eng. quorum sensing, QS) ovom patogenu omoguÄava
fleksibilnost u regulaciji ekspresije gena ukljuÄenih u virulenciju, formiranje biofilma, produkciju
sekundarnih metabolita i faktora koji imaju ulogu u zaÅ”titi od imunskog sistema domaÄina. Stoga bi
primena antivirulentne terapije zasnovane na utiÅ”avanju meÄuÄelijske komunikacije bakterija mogla
poslužiti kao obeÄavajuÄe oruÄe u kontroli infekcija izazvanih predstavnicima P. aeruginosa za koje
ne postoji adekvatna terapija trenutno dostupnim antibioticima.
U skladu sa time, predmet prouÄavanja ove teze bila je potraga za novim utiÅ”ivaÄima
meÄuÄelijske komunikacije bakterija (eng. quorum quenching, QQ) produkovanih od strane kliniÄkih
izolata koji tokom infekcija dele istu ekoloŔku niŔu sa P. aeruginosa. Analizirana je kolekcija Gramnegativnih
kliniÄkih izolata Laboratorije za molekularnu mikrobiologiju, Instituta za molekularnu
genetiku i genetiÄko inženjerstvo, Univerziteta u Beogradu sa ciljem pronalaženja sojeva koji
produkuju QQ molekule. Nakon odabira sojeva nosioca QQ fenotipa, usledila je njihova identifikacija
kao i funkcionalna karakterizacija na model sistemu kliniÄkog izolata P. aeruginosa MMA83.
Upotrebom Chromobacterium violaceum CV026 biosenzora, selektovano je 19 sojeva (od ukupno 633
analiziranih izolata) koji su ispoljavali QQ fenotip, od kojih su dva izolata Delftia sp. 11304 i
Burkholderia sp. BCC4135, na osnovu najznaÄajnije QQ aktivnosti izdvojena za detaljniju
karakterizaciju. UtvrÄeno je da Delftia sp. 11304 soj pripada Delftia tsuruhatensis vrsti, dok
Burkholderia sp. BCC4135 pripada Burkholderia cepacia vrsti sa novim sekvencnim tipom ST1485.
In silico analizom genomskih sekvenci ustanovljeno je da oba odabrana soja poseduju izuzetan
potencijal virulencije i rezistencije na antimikrobna jedinjenja. Priroda QQ molekula koje produkuju
ovi sojevi bila je razliÄita; konstatovano je da soj 11304 produkuje male molekule neproteinske prirode
(QS inhibitore, QSI), dok BCC4135 produkuje QQ enzime. Pored toga, kod BCC4135 izolata
utvrÄeno je prisustvo i QS fenomena.
Ukupni etil-acetatni ekstrakt supernatanta soja D. tsuruhatensis 11304 (QSI ekstrakt) ostvario
je znaÄajan antivirulentni potencijal na model sistemu kliniÄkog izolata P. aeruginosa MMA83...finding novel therapeutic options to combat multidrug-resistant pathogenic bacteria during the
last two decades. Given the fact that antimicrobial resistance is a complex, multifactorial phenomenon,
the solution to this problem comprises a range of approaches focused on monitoring the factors that
facilitate the emergence and spread of resistance. One of proposed strategies consists of developing
novel therapeutics that operate under different principles to the currently available antibiotics. In this
respect, antivirulence therapy has been conceived as a promising alternative to control virulence in a
pathogen-specific manner, without exerting strong selective pressure on the bacterial cells. Having in
mind that Pseudomonas aeruginosa has been considered as a leading cause of nosocomial infections
worldwide, the treatment of infections caused by this pathogen represents a major therapeutic
challenge. The pathogenic potential of P. aeruginosa has been accomplished due to a numerous innate,
acquired, and adaptive resistance mechanisms. In addition, the presence of cell-to-cell communication
system (quorum sensing, QS) allows this pathogen the flexibility in the regulation of virulence gene
expression responsible for biofilm development, production of secondary metabolites, and immuneevasive
factors. Therefore, the use of antivirulence therapy based on the silencing of bacterial
communication could serve as a promising tool in the control of infections caused by P. aeruginosa for
which there is no adequate therapy with currently available antibiotics.
Accordingly, the subject of this thesis was discovery and characterization of novel quenchers of
bacterial cell-to-cell communication produced by clinical isolates that share the same ecological niche
with P. aeruginosa during infections. A collection of Gram-negative clinical isolates from the
Laboratory for Molecular Microbiology Institute of Molecular Genetics and Genetic Engineering
University of Belgrade was analyzed in order to select the strains that produce quorum quenching
(QQ) molecules. After the selection of strains carrying QQ phenotype, the identification of QQ
molecules as well as their functional characterization on the model system of P. aeruginosa MMA83
clinical isolate was performed. Using Chromobacterium violaceum CV026 biosensor, 19 strains (out
of a total of 633 analyzed isolates) with QQ phenotype were discovered, among which two clinical
isolates Delftia sp. 11304 and Burkholderia sp. BCC4135 have been selected as strongest producers of
QQ molecules for detailed characterization. According to the genomic sequence analysis, Delftia sp.
11304 was identified as a Delftia tsuruhatensis species while Burkholderia sp. BCC4135 was unveiled
as Burkholderia cepacia with novel sequence type ST1485. In silico analysis of genomic sequences
indicated that both selected strains possess significant antimicrobial resistance and virulence potential.
The nature of the QQ molecules produced by these strains was determined as different; strain 11304
was found to produce small molecules of non-proteinaceous nature (QS inhibitors, QSI), while
BCC4135 produced QQ enzymes. Besides, the presence of the QS phenomenon was determined in
BCC4135 isolate.
The total ethyl acetate extract of the D. tsuruhatensis 11304 culture supernatant (QSI extract)
showed significant antivirulence potential on the model system of clinical isolate P. aeruginosa
MMA83..
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
RclS Sensor Kinase Modulates Virulence of Pseudomonas capeferrum
Signal transduction systems are the key players of bacterial adaptation and survival. The orthodox two-component signal transduction systems perceive diverse environmental stimuli and their regulatory response leads to cellular changes. Although rarely described, the unorthodox three-component systems are also implemented in the regulation of major bacterial behavior such as the virulence of clinically relevant pathogen P. aeruginosa. Previously, we described a novel three-component system in P. capeferrum WCS358 (RclSAR) where the sensor kinase RclS stimulates the intI1 transcription in stationary growth phase. In this study, using rclS knock-out mutant, we identified RclSAR regulon in P. capeferrum WCS358. The RNA sequencing revealed that activity of RclSAR signal transduction system is growth phase dependent with more pronounced regulatory potential in early stages of growth. Transcriptional analysis emphasized the role of RclSAR in global regulation and indicated the involvement of this system in regulation of diverse cellular activities such as RNA binding and metabolic and biocontrol processes. Importantly, phenotypic comparison of WCS358 wild type and Delta rclS mutant showed that RclS sensor kinase contributes to modulation of antibiotic resistance, production of AHLs and siderophore as well as host cell adherence and cytotoxicity. Finally, we proposed the improved model of interplay between RclSAR, RpoS and LasIR regulatory systems in P. capeferrum WCS358
Influence of amino acid substitution on the antimicrobial activity of bacteriocin lactolisterin BU
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 LBUāssecondary 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 Āµg/ml
to 0.5 Āµg/ml. An agarspot test, employing 5 Āµl 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 37Ā°C (S. aureus)
and 30Ā°C (L. lactis and L. monocytogenes).
Results: Phyre2 analysis unveiled the presence of two Ī±-helices in LBUās 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 LBUās 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
Influence of amino acid substitution on the antimicrobial activity of bacteriocin lactolisterin BU
Introduction: Lactolisterin BU (LBU) is a potent bacteriocin derived from Lactococcuslactis subsp. lactisbv. diacetylactis BGBU1-4. It exhibits antimicrobial properties against Gram-positive food spoilage andfoodborne pathogens. This research aimed to explore the impact of amino acid substitution in LBU onits antimicrobial activity by utilizing in silico prediction of LBUāssecondary structure and amino acid substitutions.Methods: The secondary structure of LBU was predicted using Phyre2 software. Five variants of LBUwere 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 Āµg/mlto 0.5 Āµg/ml. An agarspot test, employing 5 Āµl of the dilution, was conducted on three indicatorstrains:Lactococcus lactis BGMN1-596, Listeria monocytogenes ATCC19111, and Staphylococcus aureusATCC25923. The presence of inhibition zones was analyzed after overnight incubation at 37Ā°C (S. aureus)and 30Ā°C (L. lactis and L. monocytogenes).Results: Phyre2 analysis unveiled the presence of two Ī±-helices in LBUās structure. The majority of LBUvariants 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 correspondingpositions within LBUās 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
Polyphenols as Inhibitors of Antibiotic Resistant Bacteria-Mechanisms Underlying Rutin Interference with Bacterial Virulence
The rising incidence of antibiotic resistant microorganisms urges novel antimicrobials development with polyphenols as appealing potential therapeutics. We aimed to reveal the most promising polyphenols among hesperetin, hesperidin, naringenin, naringin, taxifolin, rutin, isoquercitrin, morin, chlorogenic acid, ferulic acid, p-coumaric acid, and gallic acid based on antimicrobial capacity, antibiofilm potential, and lack of cytotoxicity towards HaCaT, and to further test its antivirulence mechanisms. Although the majority of studied polyphenols were able to inhibit bacterial growth and biofilm formation, the most promising activities were observed for rutin. Further investigation proved rutin's ability to prevent/eradicate Pseudomonas aeruginosa and MRSA urinary catheter biofilms. Besides reduction of biofilm biomass, rutin antibiofilm mechanisms included reduction of cell viability, exopolysaccharide, and extracellular DNA levels. Moderate reduction of bacterial adhesion to human keratinocytes upon treatment was observed. Rutin antivirulence mechanisms included an impact on P. aeruginosa protease, pyocyanin, rhamnolipid, and elastase production and the downregulation of the lasI, lasR, rhlI, rhlR, pqsA and mvfR genes. Rutin also interfered with membrane permeability. Polyphenols could repress antibiotic resistant bacteria. Rutin has shown wide antimicrobial and antibiofilm capacity employing a range of mechanisms that might be used for the development of novel antimicrobials
Virulence traits associated with Burkholderia cenocepacia ST856 epidemic strain isolated from cystic fibrosis patients
Background: 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). Methods: 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. Results: 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). All strains expressed ability to bind to collagen and fibronectin albeit with different intensity. Representatives of ST856 exhibited gelatinase activity. ST858, ST859 and 9/11 of ST856 genotypes were positive for swimming and twitching motility whereas ST857 was non-motile. Mucoidy was demonstrated in all ST856 genotypes, ST857 was semi-mucoid, and ST858 and ST859 were non-mucoid. Molecular analysis for major virulence factors revealed that ST856 and ST857 carried the six analyzed genes, while ST858 and ST859 were negative for the llpE gene. Conclusion: Variations in virulence phenotypes in different genotypes of epidemic B. cenocepacia ST856 clone, in vitro, could be a consequence of diversification driven by pathoadaptation. Diversity of epidemic clone genotypes virulence, could be challenging for accurate diagnosis and treatment, as well as for infection control
Shotgun metagenomics reveals differences in antibiotic resistance genes among bacterial communities in Western Balkans glacial lakes sediments
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 38clusters ofattCsiteslackingintegron-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 Plav Lake. Plasmid contents within the metagenomes were similar, with proportions of contigs being plasmid-related: 1.73% for Plav Lake, 1.59% for Black Lake and 1.64% for Donje Bare Lake. The investigation showed that RNDs and mobile genetic elements content correlated with human population impact
Brevibacillus laterosporus supplementation diet modulates honey bee microbiome
Among them, bacterial and fungal pathogens Paenibacillus larvae, Melissococcus pluton, Ascosphera apis andNosema ceranae play a major impact on honey bees colonies. Thus, developing alternative prophylactic andcurative strategies are urgently needed. The use of probiotic bacteria in honey bee supplemental feeding istherefore promising to treat or prevent diseases. Brevibacillus laterosporus, Gram-positive endospore formingbacilli, is recognised as one of the promising antibacterial and antifungal agents producer.The aim of this study was to examine the short-therm effects of B. laterosporus supplemented diet on workerhoney bee microbiome.Dry spores of B. laterosporus strain BGSP11 have been administrated through a sugar syrup diet to tencolonies and a representative specimen of worker honey bees was taken before the start of the treatmentand immediately after the syrup was consumed. The microbial diversity was assessed before and after thetreatment using Illumina MiSeq sequencing platforms (ID Genomics service, Seattle, WA, USA). 16s rRNAsequencing for bacterial community profiling and fungal Internally Transcribes Spacer for mycological taxaprofiling were used. The next-generation microbiome bioinformatics platform QIIME2 v 2021.4 was used forfiltering and denoising obtained sequences, calculation of diversity metrics and taxonomy assignment. Thefeature classifier was trained using the Greengenes v 13_8 for bacterial taxa and fungal UNITE database v 8.3.The results obtained in this study indicated statisticaly significant alfa diversity between control and experimentalgroup honey bee microbiota composition. The diversity abundance was higher in control comparingto the group treated with B. laterosporus strain BGSP11 spores. There was no significant diference in Bray-Curtis distance among two groups of analysed samples. Regarding to mycological abundance, compositionwas completely different between two groups; control group had Claviceps as predominant genus, while intreated group of honey bee microbiome Metschnikowia genus was prevalent, indicating that the presence offungal pathogens in treated group is highly diminished.Book of Abstracts: Belgrade BioInformatics Conference 202
Brevibacillus laterosporus supplementation diet modulates honey bee microbiome
Among them, bacterial and fungal pathogens Paenibacillus larvae, Melissococcus pluton, Ascosphera apis and
Nosema ceranae play a major impact on honey bees colonies. Thus, developing alternative prophylactic and
curative strategies are urgently needed. The use of probiotic bacteria in honey bee supplemental feeding is
therefore promising to treat or prevent diseases. Brevibacillus laterosporus, Gram-positive endospore forming
bacilli, is recognised as one of the promising antibacterial and antifungal agents producer.
The aim of this study was to examine the short-therm effects of B. laterosporus supplemented diet on worker
honey bee microbiome.
Dry spores of B. laterosporus strain BGSP11 have been administrated through a sugar syrup diet to ten
colonies and a representative specimen of worker honey bees was taken before the start of the treatment
and immediately after the syrup was consumed. The microbial diversity was assessed before and after the
treatment using Illumina MiSeq sequencing platforms (ID Genomics service, Seattle, WA, USA). 16s rRNA
sequencing for bacterial community profiling and fungal Internally Transcribes Spacer for mycological taxa
profiling were used. The next-generation microbiome bioinformatics platform QIIME2 v 2021.4 was used for
filtering and denoising obtained sequences, calculation of diversity metrics and taxonomy assignment. The
feature classifier was trained using the Greengenes v 13_8 for bacterial taxa and fungal UNITE database v 8.3.
The results obtained in this study indicated statisticaly significant alfa diversity between control and experimental
group honey bee microbiota composition. The diversity abundance was higher in control comparing
to the group treated with B. laterosporus strain BGSP11 spores. There was no significant diference in Bray-
Curtis distance among two groups of analysed samples. Regarding to mycological abundance, composition
was completely different between two groups; control group had Claviceps as predominant genus, while in
treated group of honey bee microbiome Metschnikowia genus was prevalent, indicating that the presence of
fungal pathogens in treated group is highly diminished.Book of Abstracts: Belgrade BioInformatics Conference 202