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Harnessing the power of green and rooibos tea aqueous extracts for obtaining colored bioactive cotton and cotton/flax fabrics intended for disposable and reusable medical textiles
No full textThis study harnesses the potential of green and rooibos tea (GT and RT) aqueous extracts for obtaining colored bioactive cotton and cotton/flax fabrics with intended applications in medical textiles. The chemical characterization of the tea aqueous extracts was conducted using LC–HRMS/MS analysis, resulting in the detection of 129 bioactive compounds. GT demonstrates 2.2 times higher total phenolic content, a 14.7% lower total flavonoid content, and 3 times higher reducing power than RT. Both extracts exhibit excellent antioxidant activity (> 99.8%) and antibacterial activity (99.99%) against both tested bacteria, E. coli and S. aureus. Cotton and cotton/flax fabrics functionalized with GT or RT display outstanding antioxidant (99.63–100%) and antibacterial activity against S. aureus (90.95–99.33%), and high color strength values (5.48–11.08). The cytotoxicity assay confirmed the non-cytotoxic nature of 100% cotton fabric functionalized with GT. This sample additionally demonstrated an antibacterial reduction against E. coli and S. aureus higher than 99% and the highest release of bioactive compounds rendering it highly suitable for disposable medical textiles-wound dressings. To address the shortcomings of functionalized fabrics observed after washing, including decreased antioxidant activity (55.8–81.0%), diminished bacterial reduction, and reduced color strength values (0.80–1.36), copper-based nanoparticles (CuNPs) were biosynthesized in situ on their surfaces utilizing GT and RT aqueous extracts as reducing agents. The successful fabric decoration with CuNPs was proven by quantifying Cu2+ uptake, and characterization of the surface chemical composition and morphology of CuNPs. Colored CuNPs-decorated cotton and cotton/flax fabrics exhibited excellent antioxidant (> 98.28%) and antibacterial (99.99%) activity that remained almost unchanged after washing (94.44–98.90% and 99.99%, respectively). These fabrics are non-cytotoxic and characterized by small quantities of released bioactive compounds and Cu2+ ions into the physiological saline solution and hold promise as protective, reusable medical textiles suitable for producing gowns and drapes
Tumor necrosis factor alpha gene polymorphisms -238g˃a and -308g˃a as genetic markers for the development of alcohol related liver cirrhosis
No full textAlcohol-related liver cirrhosis (ALC) is a progressive liver disease that may result
from excessive alcohol abuse. Endotoxemia induced by ethanol consumption represents a strong stimulus
for Kupffer cells to secrete Tumor necrosis factor-α (TNF-α). TNF-α induces an inflammatory response
that often leads to chronic liver injury. It plays a key role in the pathogenesis of alcohol-related liver
disease. Our study aimed to estimate the association of ΤNF-α promoter polymorphisms at two positions
(-238G˃A and -308G˃A) with ALC susceptibility from one group of ALC patients from Serbia. A total
of 118 patients with ALC and 131 sex and age-matched healthy controls were clinically examined and
genetically tested. DNA was extracted from peripheral blood lymphocytes, and genotyping was
performed using PCR-RFLP for both promoter variants. We determined the TNF-α genotypes of each
participant by the presence of the relevant DNA fragments on agarose gel. Groups were compared using
the Chi-Square test; binary logistic regression was used to obtain odds ratios. Concerning the -238G>A
variant, a significant association between A allele carriers and risk of ALC (OR=2.36, 95% CI=1.15-
4.83; p=0.019) was observed. We found only one patient with an AA genotype, so we compared the
combined AA and GA genotypes with GG genotype. No significant differences were found in either
genotype or allelic frequencies of the –308 TNF-α gene variant between the patients and controls (allele
frequencies 15.7% vs. 13.4%, p=0.463). The -238 TNF-α –A allele was associated with a higher risk of
developing ALC in our group of patients with ALC from Serbia. This variant could represent one of the
genetic factors that confer a predisposition to develop ALC.ND B&H SYMPOSIUM OF LABORATORY GENETICISTS AND MOLECULAR BIOLOGISTS (WITH INTERNATIONAL PARTICIPATION) 10TH - 11TH MAY 2024, BANJA LUKA, BOSNIA AND HERZEGOVIN
Establishing an extracellular vesicle isolation protocol for a beneficial plant fungus
No full textIntroduction: Fusarium solani strain K (FsK) is a beneficial fungal endophyte which can help
tomato plants resist different biotic (root and foliar pathogens) and abiotic stressors
(drought, salinity). We have recently established that FsK is able to transmit sRNAs to
Nicotiana benthamiana, triggering systemic RNA silencing and DNA methylation of a
reporter gene. The mechanistic details of how the sRNAs are being transmitted still remain
elusive, but extracellular vesicles (EVs) are an emerging carrier of sRNAs.
Methods: During this study we aim to establish an effective extracellular vesicle isolation
protocol for FsK. To isolate the EVs using as starting material fungal liquid culture we applied
differential centrifugation combined with density gradient purification method. For
characterization of the isolated vesicles, the highly used NTA method was performed.
Additionally, a lipid characterization assay was also applied.
Results: After ultracentrifugation (100.000 g) the resulting pellet was separated in
iodixanol density gradient, and 10 fractions were washed and analyzed. NTA analysis
revealed the presence of two populations of particles. Starting from 250 mL of 4 days old
liquid culture of FsK, the concentration of particles was 10^10 particles/ml and 10^12
particles/ml in less dense and denser fractions, respectively. The less dense fraction
contained particles of 150 nm average size, while in denser fractions the average particle
size was 115 nm. Lipid content of isolated particles was also confirmed.
Conclusion: Using differential centrifugation combined with density gradient purification
method, high yield of particles was obtained from F.solani liquid culture. Two populations of
vesicles of different average sizes and densities were detected. Both populations had the
size range expected for EVs. Future experiments can entail the investigation of EV-associated
sRNAs and their functionality during cross-kingdom RNAi phenomena.Book of abstract: 2nd MOV E Symposium, 8-11 October 2024, Belgrade, Serbi
Prodromal Memory Impairment and Gut Dysbiosis in A53T Transgenic Model of Parkinson’s Disease – Emphasis on Social Microenvironment
No full textMost transgenic mouse models of Parkinson’s disease (PD) do not exhibit relevant
pre-motor symptoms such as cognitive decline in the visuo-spatial domain and
clinically significant dysbiosis. Additionally, given that neurobiological processes
can be altered through the brain-gut axis, co-housing healthy animals along those
genetically altered to model neurodegenerative diseases may affect both phenotypes,
potentially compromising the validity of pre-clinical research. This study aims to
address: 1) the potential of Hualpha-Syn(A53T) transgenic mice, line G2-3 as a
model of spontaneous dysbiosis and prodromal cognitive decline, and 2) the
overlooked impact of shared microenvironment on the expression of clinically
relevant indicators of prodromal PD.
Short-term spatial memory and GM composition were evaluated in Hualpha-
Syn(A53T) transgenic mice, line G2-3, which over-express human α-synuclein and
are the only model of PD that exhibits prodromal constipation. Four groups of
animals were formed: Transgenic (Tg+) mice and non-transgenic (Tg-) littermates
in mixed-group housing (MGH) and single-group housing (SGH; starting from
postnatal day 30). C57BL/6J mice were used as additional controls. Cognitive status
and memory deficits were assessed using Novel Object Recognition Test along with
Object Relocation Test in 6-month-old animals. Fecal samples were analyzed via
16S rRNA sequencing.
Results indicate that: 1) Tg+ animals exhibit GM composition similar to that
obtained in clinical settings such as an increase of bacteriodes, lactobacillus and bifidobacterium; 2) Tg+ animals exhibit memory impairment in visuo-spatial
domain regardless of housing conditions 3) Tg- exhibited memory deficits in the
spatial domain only, in MGH, while in SGH no deficits were detected; 4) β diversity
was stable regardless of housing conditions, while α diversity differed in Tg- animals
depending on the microenvironment. Certain species and genus were affected in both
Tg+ and Tg- animals.
This study reveals the potential of Hualpha-Syn(A53T) transgenic line as a model
for prodromal dysbiosis and cognitive decline in PD. Furthermore, it indicates that
the observed increase of “good bacteria” in PD-related pathology may be a
prodromal symptom of the disease, and not solely a consequence of probiotic or PD
therapy application, as it is suggested in clinical studies. Lastly, it highlights the
influence of social microenvironment on the detectability of relevant indicators in
pre-clinical settings.Book of abstracts: Belgrade Neuroscience Next Hub 2024 with international participation 24-25 May 2024. Belgrade, Serbi
MORPHOLOGICAL CHANGES OF STAPHYLOCOCCUS AUREUS AND SALMONELLA ENTERITIDIS UPON EXPOSURE TO THE EXTRACT OBTAINED FROM PLEUROTUS OSTREATUS MUSHROOM
Get PDFIn last decades, bacterial resistance to first choice
antibiotics has been drastically increasing, therefore,
the research of new antimicrobial substances
is of great importance. This rising problem
with bacterial resistance to existing antibiotics
affects not only the health care institutes but also
food plants. S. aureus and S. enteritidis pose a
number of challenges to the food industry and
cause foodborne illness in humans. In addition,
due to their favourable elemental composition,
oyster mushrooms (Pleurotus sp.) are a reservoir
of bioactive compounds that give them remarkable
antibacterial potential. P. ostreatus is of
great economic importance and is the second
most cultivated edible mushroom, therefore information
about its possible targets on bacterial
cells is of great importance for use as a dietary
supplement or medicinal purposes. The results of
the antibacterial assay showed that tested bacterial
strains were susceptible to the methanol extract of P. ostreatus (PoME), while microbicidal
activity was only detected against Gram-positive
bacteria. Scanning electron microscopy (SEM)
micrographs suggested that extract acted on
cytoplasmic membrane of S. aureus, while the
cell envelope of S. Enteritidis was the most likely
target. Natural extracts may outperform individual
bioactive compounds due to the synergistic
interaction between the metabolites, which can
enhance the effects of the individual components.
Extracts rich in antibacterials are emerging
as alternatives to synthetic antibiotics in the food
and health sectors. Among these, crude mushroom
extracts are particularly sought after for
their diverse bioactive ingredients, as they can
combat resistant strains of bacteria due to their
different targets and modes of action. PoME can
be used as an effective antimicrobial agent, suitable
for applications aiming to eradicate foodborne
pathogens, thus enhancing food safety.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 – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi
DESCRIPTION OF A NEW POTENTIAL AGGREGATION FACTOR FROM THE STREPTOCOCCUS THERMOPHILUS GENOME
Get PDFAutoaggregation, the ability to self-aggregate,
is widespread among both Gram-positive and
Gram-negative bacteria. The functional role
of aggregation is not fully understood, but it
is believed to be involved in the adaptation of
bacteria to environmental conditions (PMID:
31294207). One interesting class of compounds
responsible for the aggregation of lactic
acid bacteria is aggregation factors—surface
high-molecular-weight proteins rich in threonine
and lysine (PMID: 30027759). Recently,
our research group discovered a new strain of
Streptococcus thermophilus in the mountainous
regions of Serbia, exhibiting an aggregation
phenotype. Aggregation phenotype was confirmed
visually and using microscopy. Complete
genome of Agg+ strain was sequenced using
NGS and a gene encoding a potential aggregation
factor, which was named aggS was identified.
The predicted threonine (12.5%) and lysine
(10.5%) rich protein contains 2367 amino acids,
with an average molecular weight of 255986.63
Da. AggS also contains two cysteine residues,whereas previously well-described aggregation
factors of this type did not contain any cysteine
residues. The predicted protein includes an
N-terminal YSIRK-like signal sequence and an
LPXTG cell wall anchor domain. It has 6 Mucin
binding domain repeats alternating with 6 Mub
B2-like domain repeats. Additionally, we found a
region resembling an ice-binding domain. Given
that these bacteria endure prolonged periods of
low temperatures, it can be speculated that this
surface membrane protein also helps the bacteria
withstand freezing. The fact that the alignment
using BLASTp revealed AggS to be most
closely related to an uncharacterised protein
from the genome of Lactococcus garvieae, along
with the discovery of a transposase gene sequence
upstream of the gene, suggests that the
aggregation factor was likely acquired through
horizontal gene transfer. We plan to clone it into
a shuttle vector and investigate the aggregation
phenotype using a heterologous expression system
in Lactococcus lactis, as well as explore its
other functions.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 – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi
MEDICINAL MUSHROOM EXTRACTS ATTENUATE PSEUDOMONAS AERUGINOSA QUORUM SENSING AND VIRULENCE
Get PDFPseudomonas aeruginosa has been recognized as
a priority pathogen by World Health Organization,
due to the emergence of multidrug-resistant
(MDR) strains. Thus, new treatment options
such as antivirulence strategy is urgently needed.
This strategy is based on the disruption of quorum
sensing (QS) activity of this pathogen. The
focus of this research was to explore the anti-QS
activity of four selected medicinal mushrooms
(Lentinula edodes, Cantharellus cibarius, Trametes
versicolor and Pleurotus ostreatus) extracts on
MDR clinical isolate P. aeruginosa MMA83. Another
aim was to check their cytotoxicity on Caenorhabditis
elegans AU37 (glp-4(bn2) I; sec-1(km4).
Among three types of mushroom extracts - hot
water polysaccharide extracts (WPE), hot alkali
polysaccharide extracts (APE) and methanol
extracts (Met), APE extracts downregulated all
tested QS and virulence factors genes of P. aeruginosa
MMA83. The most prominent effect was
observed for C. cibarius APE extract, lowering
expression from 2-fold (for lasI gene) to 20-fold
for lasB gene. Extracts didn’t show cytotoxic effect
on C. elegans. The efficacy of APE extracts
in lowering the expression of QS and virulence
factors genes of P. aeruginosa MMA83 indicate
that these extracts can reduce pathogenicity of
P. aeruginosa. Also, they possess one of the desirable
biotechnology features – the absence of
cytotoxicity. Anti-QS and antivirulence effect of
APE extracts on P. aeruginosa envisages these extracts
as the promising therapeutic candidates
for the development of next-generation antivirulence
agents.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 – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi
ACINETOBACTER BAUMANNII RESISTANT TO LAST-LINE ANTIBIOTICS: AN EMERGING THREAT IN THE WESTERN BALKANS
Get PDFAcinetobacter 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 – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi
THE ROLE OF EFFLUX PUMPS IN TIGECYCLINE RESISTANCE OF ACINETOBACTER BAUMANNII ISOLATES FROM WESTERN BALKAN HOSPITALS
Get PDFThe 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.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 – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi
FROM GUT TO LAB: UNLOCKING ANTI-INFLAMMATORY POTENTIAL WITH GABA-PRODUCING BACTERIA
Get PDFPsychobiotics are live bacterial strains impacting the central nervous system, producing
neuroactive substances like GABA. GABA from
microbiota influences neural signals, affecting
neurological parameters, sleep, appetite, mood,
and cognition, traversing the intestinal barrier to bind to receptors on enteric neurons and
the vagus nerve. Lactobacillus and Bifidobacterium species can synthesize GABA from dietary
glutamate, with Lactobacillus rhamnosus shown
to reduce anxiety and depressive behavior, elevating hippocampal GABA. Limited knowledge
exists about anaerobic GABA producers, warranting further research for a comprehensive
understanding. Material for isolation comprised
fecal samples from healthy donors, with isolation conducted in an anaerobic chamber within
a maximum of 1 hour after sampling. Isolated
bacteria were identified through sequencing
the 16S rRNA gene. For bacterial cultivation, different types of media were used. PYG medium
contains hematine and vitamin K, essential supplements for the cultivation of anaerobic bacteria. All media included 0.1% L-cysteine, playing a
role in oxygen reduction, and 0.5% glutamate, a
precursor for GABA production. After identification, the presence of GABA in 8 tested bacterial
species was determined using the TLC method.
Quantification of GABA was performed using the
HPLC method. Furthermore, the positive effects
observed in Caco2 cells with induced inflammation, after treatment with certain anaerobic postbiotics producing GABA, indicate the potential
anti-inflammatory effects of these postbiotics.
The study implies anti-inflammatory effects of
anaerobic GABA producers, offering insights into the complex interplay among gut microbiota,
immune function, and mental health. Recognizing inflammation’s role in depressive symptoms,
targeting anaerobic bacteria involved in GABA
synthesis could modulate neurotransmitters and
inflammatory responses, presenting a comprehensive approach to mental well-being. Advancing research in this area contributes to a holistic
understanding of anaerobic bacteria, GABA production, gut microbiota, and mental health. This
offers avenues for novel therapeutic approaches
and enhances overall quality of life.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 – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi