Characterization of Salmonella enterica Biofilms and Antibiofilm Effect of Carvacrol and 2-Aminobenzimidazole.

Biofilm-associated foodborne Salmonella infections in poultry have become increasingly challenging for veterinarians, particularly in developing countries, and warrant thorough investigation. We assessed the biofilm-forming tendency of poultry isolates of Salmonella enterica, namely Salmonella Typhimurium (n = 23), Salmonella Infantis (n = 28), and Salmonella Heidelberg (n = 18), in nutrient-rich Rappaport-Vassiliadis Soya (RVS) peptone broth and nutrient-deficient diluted Tryptone Soya Broth (TSB). Seven of the tested isolates exhibited moderate biofilm formation in diluted TSB, whereas two showed such formation in RVS. In addition, the Congo red agar assay revealed curli and cellulose production in seven isolates. Fourteen specific biofilm-associated genes were analyzed identifying sdiA and seqA to be the most prevalent (100%), and glyA the least prevalent (69.5%). The prevalence of the genes bcsA and csgA was significantly lower in moderate and weak biofilm formers, respectively, as compared with nonbiofilm formers in RVS peptone broth. Furthermore, the compounds carvacrol and 2-aminobenzimidazole (2-ABI) effectively inhibited biofilm formation by Salmonella serovars in RVS peptone and TSB media, respectively. Whereas the antibiofilm activity of 2-ABI against Salmonella has not been reported previously, we determined its most effective concentration at 1.5 mM among tested antibiofilm treatments. These findings indicate that Salmonella strains prevalent in poultry farms have the potential to form biofilms, and the tested compounds should be further explored as supportive or alternative antimicrobials.

The role of AJB35136 and fdtA genes in biofilm formation by avian pathogenic Escherichia coli.

BACKGROUND: Infections caused by avian pathogenic Escherichia coli (APEC) result in significant economic losses in poultry industry. APEC strains are known to form biofilms in various conditions allowing them to thrive even under harsh and nutrient-deficient conditions on different surfaces, and this ability enables them to evade chemical and biological eradication methods. Despite knowing the whole genome sequences of various APEC isolates, little has been reported regarding their biofilm-associated genes. A random transposon mutant library of the wild-type APEC IMT 5155 comprising 1,300 mutants was analyzed for biofilm formation under nutrient deprived conditions using Videoscan technology coupled with fluorescence microscopy. Seven transposon mutants were found to have reproducibly and significantly altered biofilm formation and their mutated genes were identified by arbitrary PCR and DNA sequencing. The intact genes were acquired from the wild-type strain, cloned in pACYC177 plasmid and transformed into the respective altered biofilm forming transposon mutants, and the biofilm formation was checked in comparison to the wild type and mutant strains under the same conditions. RESULTS: In this study, we report seven genes i.e., nhaA, fdeC, yjhB, lysU, ecpR, AJB35136 and fdtA of APEC with significant contribution to biofilm formation. Reintroduction of AJB35136 and fdtA, reversed the altered phenotype proving that a significant role being played by these two O-antigen related genes in APEC biofilm formation. Presence of these seven genes across nonpathogenic E. coli and APEC genomes was also analyzed showing that they are more prevalent in the latter. CONCLUSIONS: The study has elucidated the role of these genes in APEC biofilm formation and compared them to adhesion expanding the knowledge and understanding of the economically significant pathogens.

Novel Avian Pathogenic Escherichia coli Genes Responsible for Adhesion to Chicken and Human Cell Lines.

Avian pathogenic Escherichia coli (APEC) is a major bacterial pathogen of commercial poultry contributing to extensive economic losses and contamination of the food chain. One of the initial steps in bacterial infection and successful colonization of the host is adhesion to the host cells. A random transposon mutant library (n = 1,300) of APEC IMT 5155 was screened phenotypically for adhesion to chicken (CHIC-8E11) and human (LoVo) intestinal epithelial cell lines. The detection and quantification of adherent bacteria were performed by a modified APEC-specific antibody staining assay using fluorescence microscopy coupled to automated VideoScan technology. Eleven mutants were found to have significantly altered adhesion to the cell lines examined. Mutated genes in these 11 "adhesion-altered mutants" were identified by arbitrary PCR and DNA sequencing. The genes were amplified from wild-type APEC IMT 5155, cloned, and transformed into the respective adhesion-altered mutants, and complementation was determined in adhesion assays. Here, we report contributions of the fdtA, rluD, yjhB, ecpR, and fdeC genes of APEC in adhesion to chicken and human intestinal cell lines. Identification of the roles of these genes in APEC pathogenesis will contribute to prevention and control of APEC infections.IMPORTANCE Avian pathogenic E. coli is not only pathogenic for commercial poultry but can also cause foodborne infections in humans utilizing the same attachment and virulence mechanisms. Our aim was to identify genes of avian pathogenic E. coli involved in adhesion to chicken and human cells in order to understand the colonization and pathogenesis of these bacteria. In contrast to the recent studies based on genotypic and bioinformatics data, we have used a combination of phenotypic and genotypic approaches for identification of novel genes contributing to adhesion in chicken and human cell lines. Identification of adhesion factors remains important, as antibodies elicited against such factors have shown potential to block colonization and ultimately prevent disease as prophylactic vaccines. Therefore, the data will augment the understanding of disease pathogenesis and ultimately in designing strategies against the infections.

Characterization of clumpy adhesion of Escherichia coli to human cells and associated factors influencing antibiotic sensitivity.

Escherichia coli intestinal infection pathotypes are characterized by distinct adhesion patterns, including the recently described clumpy adhesion phenotype. Here, we identify and characterize the genetic factors contributing to the clumpy adhesion of E. coli strain 4972. In this strain, the transcriptome and proteome of adhered bacteria were found to be distinct from planktonic bacteria in the supernatant. A total of 622 genes in the transcriptome were differentially expressed in bacteria present in clumps relative to the planktonic bacteria. Seven genes targeted for disruption had variable distribution in different pathotypes and nonpathogenic E. coli, with the pilV and spnT genes being the least frequent or absent from most groups. Deletion (Δ) of five differentially expressed genes, flgH, ffp, pilV, spnT, and yggT, affected motility, adhesion, or antibiotic stress. ΔflgH exhibited 80% decrease and ΔyggT depicted 184% increase in adhesion, and upon complementation, adhesion was significantly reduced to 13%. ΔflgH lost motility and was regenerated when complemented, whereas Δffp had significantly increased motility, and reintroduction of the same gene reduced it to the wild-type level. The clumps produced by Δffp and ΔspnT were more resistant and protected the bacteria, with ΔspnT showing the best clump formation in terms of ampicillin stress protection. ΔyggT had the lowest tolerance to gentamicin, where the antibiotic stress completely eliminated the bacteria. Overall, we were able to investigate the influence of clump formation on cell surface adhesion and antimicrobial tolerance, with the contribution of several factors crucial to clump formation on susceptibility to the selected antibiotics. IMPORTANCE: The study explores a biofilm-like clumpy adhesion phenotype in Escherichia coli, along with various factors and implications for antibiotic susceptibility. The phenotype permitted the bacteria to survive the onslaught of high antibiotic concentrations. Profiles of the transcriptome and proteome allowed the differentiation between adhered bacteria in clumps and planktonic bacteria in the supernatant. The deletion mutants of genes differentially expressed between adhered and planktonic bacteria, i.e., flgH, ffp, pilV, spnT, and yggT, and respective complementations in trans cemented their roles in multiple capacities. ffp, an uncharacterized gene, is involved in motility and resistance to ampicillin in a clumpy state. The work also affirms for the first time the role of the yggT gene in adhesion and its involvement in susceptibility against another aminoglycoside antibiotic, i.e., gentamicin. Overall, the study contributes to the mechanisms of biofilm-like adhesion phenotype and understanding of the antimicrobial therapy failures and infections of E. coli.

MicroRNA biomarkers as next-generation diagnostic tools for neurodegenerative diseases: a comprehensive review.

Neurodegenerative diseases (NDs) are characterized by abnormalities within neurons of the brain or spinal cord that gradually lose function, eventually leading to cell death. Upon examination of affected tissue, pathological changes reveal a loss of synapses, misfolded proteins, and activation of immune cells-all indicative of disease progression-before severe clinical symptoms become apparent. Early detection of NDs is crucial for potentially administering targeted medications that may delay disease advancement. Given their complex pathophysiological features and diverse clinical symptoms, there is a pressing need for sensitive and effective diagnostic methods for NDs. Biomarkers such as microRNAs (miRNAs) have been identified as potential tools for detecting these diseases. We explore the pivotal role of miRNAs in the context of NDs, focusing on Alzheimer's disease, Parkinson's disease, Multiple sclerosis, Huntington's disease, and Amyotrophic Lateral Sclerosis. The review delves into the intricate relationship between aging and NDs, highlighting structural and functional alterations in the aging brain and their implications for disease development. It elucidates how miRNAs and RNA-binding proteins are implicated in the pathogenesis of NDs and underscores the importance of investigating their expression and function in aging. Significantly, miRNAs exert substantial influence on post-translational modifications (PTMs), impacting not just the nervous system but a wide array of tissues and cell types as well. Specific miRNAs have been found to target proteins involved in ubiquitination or de-ubiquitination processes, which play a significant role in regulating protein function and stability. We discuss the link between miRNA, PTM, and NDs. Additionally, the review discusses the significance of miRNAs as biomarkers for early disease detection, offering insights into diagnostic strategies.

A multiparametric fluorescence assay for screening aptamer-protein interactions based on microbeads.

For improving aptamer-ligand binding we have developed a screening system that defines optimal binding buffer composition. Using multiplex assays, one buffer system is needed which guarantees the specific binding of all aptamers. We investigated nine peer-reviewed DNA aptamers. Non-specific binding of aptamers is an obstacle. To address this, we investigated 16 proteins as specificity controls bound covalently to encoded microbeads in a multiplex assay. Increasing the NaCl concentration decreased the binding for all aptamers. Changing pH values by one unit higher or lower did not influence the aptamer binding significantly. However, pH < 5 led to non-specific binding for all aptamers. The PfLDH-aptamer selected in the absence of divalent cations exhibited doubling of its binding signal by the addition of Ca2+ and Mg2+. We confirmed Ca2+ and Mg2+ dependency of the aptamers for streptavidin and thrombin by observing a 90% and 50% binding decrease, respectively. We also achieved a doubling of binding for the streptavidin aptamer when replacing Ca2+ and Mg2+ by Mn2+. A buffer suitable for all aptamers can have considerable variations in pH or ionic strength, but divalent cations (Ca2+, Mg2+, Mn2+) are essential.

Whatever makes them stick - Adhesins of avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is associated with extraintestinal infections and the development of colibacillosis, causing high mortality in farm birds and extensive losses in the poultry industry worldwide. The virulence of APEC is a complex phenomenon associated with numerous mechanisms involving a variety of extracellular and intracellular structures to overcome host barriers. Initial bacterial attachment or adhesion to host cells is vital to bacterial pathogenesis and is determined by various adhesins. These proteins protect pathogens against possible host defense mechanisms, enabling the effective use of other virulence attributes. Considering this property, the current review provides a systematic and in-depth analysis of the latest information on adhesins analyzed in APEC strains. This review discusses in detail each of the adhesin types, such as fimbrial chaperone-usher, fimbrial curli, nonfimbrial and atypical adhesins, and their components, presenting an opportunity to gain a better understanding of different adhesins and mechanisms employed in pathogenesis. Additionally, the article scrutinizes and notes missing information and potential studies that need to be undertaken to develop a complete understanding of APEC adhesion.

Genome placement of alpha-haemolysin cluster is associated with alpha-haemolysin sequence variation, adhesin and iron acquisition factor profile of Escherichia coli.

Since the discovery of haemolysis, many studies focused on a deeper understanding of this phenotype in Escherichia coli and its association with other virulence genes, diseases and pathogenic attributes/functions in the host. Our virulence-associated factor profiling and genome-wide association analysis of genomes of haemolytic and nonhaemolytic E. coli unveiled high prevalence of adhesins, iron acquisition genes and toxins in haemolytic bacteria. In the case of fimbriae with high prevalence, we analysed sequence variation of FimH, EcpD and CsgA, and showed that different adhesin variants were present in the analysed groups, indicating altered adhesive capabilities of haemolytic and nonhaemolytic E. coli. Analysis of over 1000 haemolytic E. coli genomes revealed that they are pathotypically, genetically and antigenically diverse, but their adhesin and iron acquisition repertoire is associated with genome placement of hlyCABD cluster. Haemolytic E. coli with chromosome-encoded alpha-haemolysin had high frequency of P, S, Auf fimbriae and multiple iron acquisition systems such as aerobactin, yersiniabactin, salmochelin, Fec, Sit, Bfd and hemin uptake systems. Haemolytic E. coli with plasmid-encoded alpha-haemolysin had similar adhesin profile to nonpathogenic E. coli, with high prevalence of Stg, Yra, Ygi, Ycb, Ybg, Ycf, Sfm, F9 fimbriae, Paa, Lda, intimin and type 3 secretion system encoding genes. Analysis of HlyCABD sequence variation revealed presence of variants associated with genome placement and pathotype.

Investigation of Commensal Escherichia coli Populations of Cormorant Hatchlings in the Absence of Anthropogenic Impacts in Remote Areas of West Mongolia.

To increase our understanding of bacterial intestinal colonization in animal populations lacking substantial anthropogenic influence we studied the diversity of E. coli in cormorants from the pristine West-Mongolian steppe. E. coli were isolated from individual birds of two cormorant colonies located on small islands in lakes at least 100 km away from human settlements. Diversity of the isolates was studied using pulsed-field gel electrophoresis (PFGE). 137 isolates of cormorant colony-1 and 75 isolates of cormorant colony-2 resulted in 60 and 33 PFGE types, respectively. Representative strains of each PFGE type were analyzed via PCR in terms of phylogroups and extraintestinal virulence-associated genes (exVAGs). Bacterial adhesion to the chicken intestinal cell line CHIC-8E11 and antimicrobial resistance was also determined. Most isolates belonged to phylogroup B1 (68.3%) followed by B2 and E with B2 harboring the highest total number of exVAGs per isolate. Unexpectedly, a PFGE type with relatively few exVAGs displayed the highest isolation frequency, also showing a high adhesion rate. Comparative analysis of exVAGs to other E. coli populations of wildlife origin revealed that the secreted autotransporter toxin encoding sat gene was only present in cormorants. Overall, E. coli in cormorants maintained a high diversity under minimal anthropogenic influences, which likely enables intestinal colonization.

Genomic and Phenotypic Analysis of an ESBL-Producing E. coli ST1159 Clonal Lineage From Wild Birds in Mongolia.

BACKGROUND: In addition to the broad dissemination of pathogenic extended-spectrum beta-lactamase (ESBL)-producing Escherichia (E.) coli in human and veterinary medicine and the community, their occurrence in wildlife and the environment is a growing concern. Wild birds in particular often carry clinically relevant ESBL-producing E. coli. OBJECTIVES: We analyzed ESBL-producing and non-ESBL-producing E. coli obtained from wild birds in Mongolia to identify phylogenetic and functional characteristics that would explain the predominance of a particular E. coli clonal lineage in this area. METHODS: We investigated ESBL-producing E. coli using whole-genome sequencing and phylogenetics to describe the population structure, resistance and virulence features and performed phenotypic experiments like biofilm formation and adhesion to epithelial cells. We compared the phenotypic characteristics to non-ESBL-producing E. coli from the same background (Mongolian wild birds) and genomic results to publicly available genomes. RESULTS AND CONCLUSION: We found ESBL-producing E. coli sequence type (ST) 1159 among wild birds in Mongolia. This clonal lineage carried virulence features typical for extra-intestinal pathogenic or enterotoxigenic E. coli. Comparative functional experiments suggested no burden of resistance in the ST1159 isolates, which is despite their carriage of ESBL-plasmids. Wild birds will likely disseminate these antibiotic-resistant pathogens further during migration.

Pb-induced phytotoxicity in para grass (Brachiaria mutica) and Castorbean (Ricinus communis L.): Antioxidant and ultrastructural studies.

Hydroponics experiment was conducted to investigate the effects of different levels of Pb on Para Grass (Brachiaria mutica) and Castorbean (Ricinus communis L). Generally, Para Grass exhibited higher tolerance to excessive concentrations of Pb in nutrient solution, whereas a consistent decline was observed in growth of Castorbean plants exposed to similar Pb levels. Malondialdehyde (MDA) and H2O2 contents exhibited contrasting results with a general decrease in Para Grass and a linear increase in case of Castorbean. In both species a decrease was noticed in the activities of superoxide dismutase (SOD) and guaiacol peroxidase (G-POD) while catalase (CAT) activity was significantly increased. Ultrastructural studies revealed increased starch grains and adversely affected thylakoid membranes in chloroplasts of leaf cells of plants treated with 500 µM Pb. Photosynthetic parameters such as CO2 assimilation rate, stomatal conductance (gs) and transpiration rate (E) decreased in both plant species under different levels of Pb. Maximum concentrations of Pb in shoots of Para Grass and Castorbean were 1.29 and 0.352 g kg-1, respectively while in roots maximum values were 8.88 and 49.86 g kg-1, respectively. The high concentrations of Pb (about 5%) in the roots of Castorbean plants suggest its possible role in the phytoremediation/rhizofiltration of Pb contaminated water.