NEW APPROACHES IN THE TREATMENT OF CHRONIC BACTERIAL INFECTIONS

The rapid emergence and spread of multidrug- resistant pathogens present a global healthcare challenge. One common cause of resistance and/or tolerance to antibiotics is biofilms, a complex communities of bacteria embedded in a self-produced matrix. Biofilm formation and maturation are regulated by quorum sensing, a cell density-dependent communication system that relies on the synthesis, diffusion, and detection of small signaling molecules - autoinducers (AIs). Quorum quenching (QQ) enzymes that cut Ais emerged as a promising strategy for persistent bacterial infections. However, a significant drawback for the use of QQ enzymes as therapeutics is their poor stability and efficacy in vivo. Since one of the major health issues linked to biofilm development is persistent wound infections, our goal was to improve enzyme properties by immobilizing it on a natural biopolymer to make it suitable for use as a wound dressing. The best candidate for immobilization was YtnP lactonase from Bacillus paralicheniformis ZP1, as in concentrations higher than 25 μg/mL it improved the survival of Pseudomonas aeruginosa PAO1-infected zebrafish, rescuing 80% of embryos. When combined with tobramycin or gentamicin, the survival rate of zebrafish embryos increased to 100%. Purified YtnP lactonase at a concentration of 1 mg was immobilized on 10 mg of polymer disks by crosslinking with glutaraldehyde. Specific modifications of the polymer were also made to eliminate the use of glutaraldehyde, which is a skin irritant. In in vivo experiments on a murine chronic wound model, immobilized enzyme inhibited biofilm development, cleared already formed biofilms, and overall improved wound healing. These results provide a foundation for the development of advanced wound dressings that will prevent infection development in wounds and enable proper therapy for infected chronic wounds.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

Development of pcr-based identification of salmonella enterica serovars

The aim of the study was to evaluate and adapt the PCR-based protocol that utilizes the developed serotype-specific primers to identify Salmonella enterica species and its serotypes that are most frequently isolated from poultry samples in Vojvodina. Using the slide agglutination test, 64 and 33 out of 107 Salmonella isolates were identified as S. Infantis and S. Enteritidis, respectively, while ten isolates were identified as eight different Salmonella serovars. Using the same isolates, presence of 993-bp (bcfC gene), 636-bp (steB gene) and 293-bp (sdf locus) amplicons in multiplex PCR unambiguously identified 31 isolates as S. Enteritidis. Two isolates identified as Enteritidis in slide agglutination test were not identified as such in PCR-based approach since they both were missing 293bp long PCR product. Thirty-nine isolates produced a 727-bp amplicon in the specific simplex PCR, and thus were identified as S. Infantis. The greatest discrepancy in comparison to the results of conventional serotyping has been observed in the case of S. Infantis, since 25 more isolates were noted as S. Infantis by conventional serotyping. Seven isolates, with unexpected PCR profiles stayed unidentified by molecular typing, although they were serotyped as S. Typhimurium (1) and S. Infantis (6). S. Gallinarum serovar has to be additionally confirmed, since it shares the same PCR profile with S. Livingstone. Clearly, PCR-based identification has to be thoroughly checked, verified and adapted if it is to be applied as the routine identification protocol

Glycosylation and pH stability of penicillin G acylase from providencia rettgeri produced in Pichia pastoris

Penicilin G acilaza (PAC) je jedan od najšire korišćenih enzima u industrijskoj sintezi polusintetskih antibiotika. U ovom radu dobijeni nivo ekspresije PAC gena iz Providencia rettgeri u ekspresionom sistemu Pichia pastoris iznosio je 2.7 U/ml. Rekombinantni enzim je prečišćen i određen je njegov glikozilacioni status. Nađeno je da osim što su obe subjedinice enzima (α i β) N-glikozilovane, β subjedinica sadrži još i O-glikane. Takođe je ustanovljeno da je rekombinantna PACP. rett. stabilna u širokom pH opsegu što ju je, zajedno sa predhodno ustanovljenom visokom termostabilnošću, učinilo izuzetno privlačnim biokatalizatorom sa industrijske tačke gledišta.Penicillin G acylase (PAC) is one of the most widely used enzymes in industrial synthesis of semi-synthetic antibiotics. The Providencia rettgeri pac gene was expressed to a level of 2.7 U/ml using the Pichia pastoris expression system. The recombinant enzyme was purified and its glycosylation status was determined. It was found that both subunits (α and β) of the enzyme were N-glycosylated, while the β-subunit also contained O-glycans. It was also observed that rPACP.rett. was stable in a wide range of pH, which, in addition to the previously proved high thermostability, makes it an attractive biocatalyst from an industrial point of view

Transcriptome analysis of Pseudomonas aeruginosa after MhqO dioxygenase treatment

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe chronic infections due to its exceptional ability to form a biofilm. Regulation of biofilm formation is very sophisticated and involves multiple bacterial systems and regulatory pathways. We found an enzyme MhqO dioxygenase from Bacillus paralicheniformis ZP1, which was effective in the inhibition of biofilm formation and disruption of mature biofilm of P. aeruginosa. Our results suggest that MhqO exerts its effect at the adhesion level, preventing cells from attaching to the surface. We have also shown that the enzyme stimulates the rhamnolipids synthesis. To elucidate the mechanism of enzyme action, we analyzed the transcriptome of the P. aeruginosa PAO1 strain treated with MhqO. Since cell adhesion occurs at the beginning of the stationary phase growth, the PAO1 strain was treated with MhqO for four hours, followed by total RNA isolation and cDNA synthesis. Transcriptome sequencing was performed by Illumina NovaSeq 6000 and data were analyzed by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). Obtained data showed that 122 genes were up-regulated, 41 genes were down-regulated, and the expression of 5947 genes was not changed. Five genes whose expression was altered are directly related to biofilm formation. MhqO increased the expression of the RsmA post-transcriptional regulator in P. aeruginosa. Transcriptome data revealed that pili IV biosynthesis genes were up-regulated, which is in accordance with literature data that RsmA positively regulates these genes. The inhibition of cells’ attachment to the surface could be explained by these results. In addition, RsmA positively regulates rhamnolipid production but negatively regulates biofilm matrix synthesis, which was supported by expression levels in the sequenced transcriptome. Data obtained from transcriptome analysis suggest that P. aeruginosa treated with MhqO dioxygenase should be more sensitive to oxidative and osmotic stress, as well as to beta-lactam antibiotics. Our further investigations should confirm these effects at the phenotypic level as well.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Β-glucosidase b from microbacterium sp. Bg28 as a biofilm control agent In food processing environment

About one in ten people contract a foodborne illness within a year. Children under the age of five are the most affected, with 125,000 deaths each year. Many of the foodborne illness outbreaks can be linked to the presence of biofilms in the food industry, and Salmonella enteritidis is an extremely important foodborne pathogen that thrives in these conditions. It has been shown that biofilms can be resistant to physical and chemical treatments used in cleaning and disinfection procedures in food processing. The problem with using more aggressive disinfectants is that they often violate food safety regulations. The use of enzymes which degrade biofilm matrix structural components should facilitate current disinfection procedures and not compromise food safety. In this study, the anti-biofilm activity of recombinantly expressed β-glucosidase B and its potential use as a protective agent to control Salmonella biofilm formation is investigated. The putative target of this enzyme is cellulose, the structural component of the Salmonella biofilm matrix. β-Glucosidase B deriving from the environmental strain Microbacterium sp. BG28 was heterologously expressed in Escherichia coli and successfully purified by affinity chromatography. The anti-biofilm activity of the enzyme was evaluated in in vitro assays using various clinical isolates of S. enteritidis. The toxicity of the enzyme was studied in Caenorhabditis elegans. β-glucosidase B effectively inhibited the formation of Salmonella biofilms grown in a temperature range of 8°C to 37°C, achieving 50% inhibition at concentrations of 100μg/ml. Biochemical characterization showed that the optimal pH activity of the enzyme is between 6 and 7, with the highest activity observed at temperatures between 37°C and 47°C. The absence of toxicity and other presented results indicate that beta-glucosidase B can be used in biofilm control in the food industry.Book of abstracts: International Conference of Biochemists and Molecular Biologists in Bosnia and Herzegovina - ABMBBIH May, 202

Potential of environmental Serratia from herbicide treated soil for pathogenicity in humans

Soils contain microorganisms capable of causing diseases in humans, either as opportunistic or as obligate pathogens. The application of herbicides increases the prevalence of antibiotic resistance genes and mobile genetic elements, which expands the competitive niche of pathogenic microbes. This implies that herbicides could impose selective pressure driving the spread of antibiotic resistance and therefore making heavily herbicide treated agricultural soil a possible reservoir of multiresistant opportunistic human pathogens. From the soil exposed to chlorinated aromatic herbicides S-metolachlor and isoxaflutole, semi-persistent selective herbicides widely used in European Union and Serbia we isolated bacteria able to grow on herbicide as the sole carbon source. Bacterial isolates were assessed for various virulence traits and resistance to antibiotics in order to identify the strains with high pathogenic potential

Functional Modification of Cellulose Acetate Microfiltration Membranes by Supercritical Solvent Impregnation

This study investigates the modification of commercial cellulose acetate microfiltration membranes by supercritical solvent impregnation with thymol to provide them with antibacterial properties. The impregnation process was conducted in a batch mode, and the effect of pressure and processing time on thymol loading was followed. The impact of the modification on the membrane's microstructure was analyzed using scanning electron and ion-beam microscopy, and membranes' functionality was tested in a cross-flow filtration system. The antibiofilm properties of the obtained materials were studied against Staphyloccocus aureus and Pseudomonas aeruginosa, while membranes' blocking in contact with bacteria was examined for S. aureus and Escherichia coli. The results revealed a fast impregnation process with high thymol loadings achievable after just 0.5 h at 15 MPa and 20 MPa. The presence of 20% of thymol provided strong antibiofilm properties against the tested strains without affecting the membrane's functionality. The study showed that these strong antibacterial properties could be implemented to the commercial membranes' defined polymeric structure in a short and environmentally friendly process

In silico pre-selection of β-glucosidase gene for heterologous recombinant expression

Biofilms are ubiquitous in nature, and the food industry is vulnerable to the risks posed by biofilm formation. Not only do they interfere with the food production process, but they also pose a public health threat. However, complete elimination of biofilms on food and food contact surfaces cannot be achieved by conventional methods (cleaning and disinfection) alone. New biofilm control strategies must be developed to prevent its formation and/or persistence. Novel approaches may be based on enzymes that depolymerize components of the biofilm matrix, making bacterial cells accessible to antimicrobial agents. Environmental microorganisms are an inexhaustible source of new enzymes. In Salmonella Enteritidis and Escherichia coli, known foodborne pathogens, cellulose is an important component of the biofilm matrix, so our isolates from untapped environments were tested for cellulolytic activity. Of the more than 70 isolates examined, isolate BG28 was selected as the most promising. Its genome was sequenced, annotated, and it was identified as Gram-positive Microbacterium sp. Genome mining revealed the presence of four complete genes for different β-glucosidases, one of three enzyme types of cellulase complexes. To select the best candidate for heterologous expression DeepTMHMM, ProtParam, and SoluProt were used to predict the presence/absence of signal peptide and transmembrane domains, instability index, aliphatic index, hydrophilicity, and soluble expression in E. coli. Based on the prediction results, the gene annotated as β-glucosidase B was selected for recombinant expression. In addition, I-TASSER was used to model the tertiary structure of the selected enzyme. The β-glucosidase B was recombinantly expressed, purified, and tested for its anti-biofilm activity. It was active and showed a 50% inhibitory effect on S. Enteritidis and E. coli biofilm formation at a concentration of 100 μg/ml. To further evaluate this in silico approach in the preselection of candidate enzymes for recombinant expression and purification, we will use it to identify other enzymes of the cellulase complex.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Limited aromatic pathway genes diversity amongst aromatic compound degrading soil bacterial isolates

Identifikacija i karakterizacija novih gena koji pripadaju putevima mikrobiološke razgradnje aromatičnih jedinjenja je od velikog značaja, jer su se pokazali kao izuzetno dobri biokatalizatori. U ovoj studiji, korišćenjem PCR metodologije, analizirano je prisustvo pet različitih gena iz biodegradativnog puta aromatičnih jedinjenja među 19 sredinskih izolata sa sposobnošću razgradnje širokog spektra aromatičnih jedinjenja. U slučaju 4-oksalokrotonat tautomeraze i toluen dioksigenaze, koji su detektovani kod većine sredinskih izolata, sekvence fragmenata su ukazivale na veoma ograničen diverzitet ova dva gena i visoku homologiju sa već poznatim sekvencama opisanim kod vrsta roda Pseudomonas. Korišćenjem degenerisanih prajmera konstruisanih na osnovu poznatih katehol-i naftalendioksigenaznih gena vrlo mali broj fragmenata je amplifikovan kod sredinskih izolata. Samo dve katehol 2,3-dioksigenaze iz dva izolata roda Bacillus su sekvenciranjem ukazale na različitost u odnosu na poznate sekvence, a pokazale međusobnu sličnost od 80-90%. Potencijalno tri nove katehol 1,2-dioksigenaze su identifikovane kod Bacillus sp. TN102, Gordonia sp. TN103 i Rhodococcus sp. TN112. Visok stepen homologije tautomeraza i toluen dioksigenaza među sredinskim izolatima izolovanim iz zagađene sredine ukazuje na horizontalni transfer gena, dok je ograničen uspeh u detektovanju preostala tri gena ukazao na potencijal da se među ovim izolatima mogu naći nove varijante gena iz puteva razgradnje aromatičnih jedinjenja.Identification and characterization of novel genes belonging to microbial aromatic biodegradation pathway is of great importance as they have been proven versatile biocatalysts. In this study, the selection of 19 environmental bacterial isolates capable to degrade a wide range of aromatic compounds has been screened for the presence of five genes from the lower and the upper aromatic biodegradation pathway using PCR methodology. In the case of 4-oxalocrotonate tautomerase and toluene dioxygenases, although present in the most of environmental isolates, very limited diversity of the genes has been encountered. Highly conserved sequences of these genes in environmental samples revealed high homology with gene sequences of the characterized corresponding genes from Pseudomonas putida species. The screen using degenerate primers based on known catechol-and naphthalene dioxygenases sequences resulted in a limited number of amplified fragments. Only two catechol 2,3-dioxygenase from two Bacillus isolates were amplified and showed no significant similarities with dioxygenases from characterized organisms, but 80-90% identities with partial catechol 2,3-dioxygenase sequences from uncultured organisms. Potentially three novel catechol 1,2-dioxygenases were identified from Bacillus sp. TN102, Gordonia sp. TN103 and Rhodococcus sp. TN112. Highly homologous tautomerase and toluene dioxygenases amongst environmental samples isolated from the contaminated environment suggested horizontal gene transfer while limited success in PCR detection of the other three genes indicates that these isolates may still be a source of novel genes

Anti-Virulence Potential and In Vivo Toxicity of Persicaria maculosa and Bistorta officinalis Extracts

Many traditional remedies represent potential candidates for integration with modern medical practice, but credible data on their activities are often scarce. For the first time, the anti-virulence potential and the safety for human use of the ethanol extracts of two medicinal plants, Persicaria maculosa (PEM) and Bistorta officinalis (BIO), have been addressed. Ethanol extracts of both plants exhibited anti-virulence activity against the medically important opportunistic pathogen Pseudomonas aeruginosa. At the subinhibitory concentration of 50 mu g/mL, the extracts demonstrated a maximal inhibitory effect (approx. 50%) against biofilm formation, the highest reduction of pyocyanin production (47% for PEM and 59% for BIO) and completely halted the swarming motility of P. aeruginosa. Both extracts demonstrated better anti-quorum sensing and antibiofilm activities, and a better ability to interfere with LasR receptor, than the tested dominant extracts' constituents. The bioactive concentrations of the extracts were not toxic in the zebrafish model system. This study represents an initial step towards the integration of P. maculosa and B. officinalis for use in the treatment of Pseudomonas infections