Biology subjects

Bouras, G.

Publications and source records attributed to Bouras, G..

6 recordsLinked to original sources

bioRxiv2023

Bacteriophage in combination with ciprofloxacin against Pseudomonas aeruginosa infections in diabetic foot ulcer patients

BackgroundIn diabetic foot ulcer (DFU) patients, Pseudomonas aeruginosa (P. aeruginosa) infections are linked to poor wound healing. The ineffectiveness of antibiotics against these infections promotes the emergence of multidrug-resistant (MDR) strains. Bacteriophage (phage) therapy has recently gained popularity as an alternative to antibiotics. MethodologyBacterial and viral swabs and tissue were obtained from DFU infections (DFI). Bacteria were cultured followed by MALDI-TOF MS for identification. 16S rRNA long-read sequencing was used to identify the microbiota. Bacteriophages were isolated and underwent transmission electron microscopy, genomic sequencing, and stability testing. The antimicrobial activity of phages alone and in combination with ciprofloxacin against P. aeruginosa planktonic cells and biofilm grown in vitro and in ex vivo tissue was tested by measuring the optical density (OD), crystal violet assays and live/dead staining with visualisation using confocal scanning laser microscopy respectively. ResultsA total of 34 DFI patients were recruited from which microbiota were analysed for 25 patients. P. aeruginosa was the most prevalent pathogen cultured and was one of the top 6 most prevalent and abundant species in the microbiota analysis. Phage APTC-PA18 was isolated from DFIs, belonged to the myoviridae family and was strictly lytic. PA18 was stable between 4 and 70 degrees Celsius and between pH 3 and 11. Seven of eight P. aeruginosa clinical isolates were sensitive to APTC-PA18, and when APTC-PA18 was combined with ciprofloxacin against planktonic and biofilm of P. aeruginosa, synergistic effects were observed in vitro and in DFI tissue samples. ConclusionPhage APTC-PA18, when combined with ciprofloxacin, has the ability to kill P. aeruginosa clinical isolates both in vitro and ex vivo and is a promising treatment option for P. aeruginosa infections in DFUs.

microbiology↗

bioRxiv2023

Prophage acquisition by Staphylococcus aureus contributes to the expansion of Staphylococcal immune evasion

Staphylococcus aureus colonizes 30% of the human population, but only a few clones cause severe infections. S. aureus virulence varies and partly depends on the presence of prophages, viral DNA embedded in the S. aureus core genome, such as hlb-converting prophage ({phi}Sa3int). Human-adapted S. aureus often harbours a {phi}Sa3int group of prophages preferentially integrated into their {beta}-hemolysin (hlb) gene that encodes human immune evasion cluster (IEC) genes. Exotoxins and immune modulatory molecules encoded by this prophage can inhibit human innate immunity increasing S. aureus pathogenicity. This study aims to investigate the genomic and phenotypic plasticity of S. aureus and changes in its extracellular proteome after the acquisition of {phi}Sa3int prophage. To achieve this, we used S. aureus strains isolated from the sinus cavities of a patient with severe chronic rhinosinusitis (CRS) at two different time points (S. aureus SA222 and S. aureus SA333) and hybrid sequenced the strains using short-read Illumina and long-read Oxford nanopore technology. In silico analysis showed the presence of a {phi}Sa3int prophage in the later isolate but not in the earlier isolate while most of the core genes remained identical. Using mitomycin C, we induced the {phi}Sa3int prophage, and transduced it into the Sa3int-prophage-free SA222 isolate to obtain a laboratory generated double lysogen. We confirmed the successful lysogenisation with culture methods (spot assay, blood agar) and also by sequencing. We compared growth kinetics, biofilm biomass and metabolic activity between parent and the lysogen by establishing growth curves, crystal violet and resazurin assays. Exoproteins were identified and quantified using mass spectrophotometry. Integration of {phi}Sa3int prophage in SA222 down-regulated the beta-hemolysin expression of the lysogen. In silico analysis of the S. aureus genome confirmed the insertion of a [~]43.8 kb {phi}Sa3int prophage into hlb gene. Insertion of prophage DNA did not alter the growth kinetics, biofilm formation, adhesion to primary human nasal epithelial cells and the metabolic activity in a biofilm. However, the acquisition of {phi}Sa3int prophage significantly changed the expression of various secreted proteins, both bacterial and prophage-encoded. Altogether, thirty-eight exoproteins were significantly differentially regulated in the laboratory created lysogen, compared to its recipient strain SA222. Among these proteins, there was significant upregulation of 21 exoproteins (55.3 %) including staphylokinase (sak), SCIN (scn), and intercellular adhesion protein B (icaB) and downregulation of 17 exoproteins (44.7 %), including {beta}-hemolysin (hlb/sph) and outer membrane porin (phoE). Most of the upregulated proteins were involved in immunomodulation that help S. aureus escape human innate immunity and help cause chronic infection. These findings may contribute to the development of novel approaches to render S. aureus susceptible to the immune response by blocking prophage-associated defence mechanisms. HighlightsO_LIA {phi}Sa3int prophage preferentially integrates into the {beta}-haemolysin gene (hlb) gene thereby disrupting the beta-hemolysin function. C_LIO_LIA [~]43.8 kb {phi}Sa3int prophage acquisition by S. aureus has no impact on its growth kinetics, biofilm formation and adhesion to primary human nasal epithelial cells (HNECs). C_LIO_LIThe presence of a {phi}Sa3int group prophage likely enhances Staphylococcus aureus human immune evasion capability as the prophage encodes a complete set of immune evasion cluster (IEC) genes. C_LIO_LITargeted identification of virulence factors in addition to species and strain identification may lead to better-personalized therapy as not all S. aureus carry the same virulence genes. C_LI

microbiology↗

bioRxiv2023

Phables: from fragmented assemblies to high-quality bacteriophage genomes

MotivationMicrobial communities influence both human health and different environments. Viruses infecting bacteria, known as bacteriophages or phages, play a key role in modulating bacterial communities within environments. High-quality phage genome sequences are essential for advancing our understanding of phage biology, enabling comparative genomics studies, and developing phage-based diagnostic tools. Most available viral identification tools consider individual sequences to determine whether they are of viral origin. As a result of the challenges in viral assembly, fragmentation of genomes can occur, leading to the need for new approaches in viral identification. Therefore, the identification and characterisation of novel phages remain a challenge. ResultsWe introduce Phables, a new computational method to resolve phage genomes from fragmented viral metagenome assemblies. Phables identifies phage-like components in the assembly graph, models each component as a flow network, and uses graph algorithms and flow decomposition techniques to identify genomic paths. Experimental results of viral metagenomic samples obtained from different environments show that Phables recovers on average over 49% more high-quality phage genomes compared to existing viral identification tools. Furthermore, Phables can resolve variant phage genomes with over 99% average nucleotide identity, a distinction that existing tools are unable to make. Availability and ImplementationPhables is available on GitHub at https://github.com/Vini2/phables. Contactvijini.mallawaarachchi@flinders.edu.au

bioinformatics↗

bioRxiv2023

Staphylococcus aureus biofilm secreted factors cause mucosal damage, mast cell infiltration and goblet cell hyperplasia in a rat rhinosinusitis model

Chronic Rhinosinusitis (CRS) is an inflammatory condition of the paranasal sinus mucosa. Despite being a common health issue, the exact cause of CRS is yet to be understood. However, research suggests that Staphylococcus aureus, particularly in the biofilm form, drives the disease. This study aimed to investigate the impact of long-term exposure to secreted factors of Staphylococcus aureus biofilm (SABSF), harvested from clinical isolates of non-CRS carriers and CRS patients, on the nasal mucosa in a rat model. Wistar rats were randomised (n=5/group) to receive daily intranasal instillations of 40 L (200 g/L) SABSF for 28 days or vehicle control with S. aureus isolated from the sinuses of a non-CRS carrier, a type 2 endotype CRS with nasal polyps (CRSwNP) patient, and a non-type 2 endotype CRS without nasal polyps (CRSsNP) patient. The sinonasal samples of the rats were then analysed through histopathology and transcriptome profiling. The results showed that all three intervention groups displayed significant lymphocytic infiltration (p[≤]0.05). However, only the SABSF collected from the CRSwNP patient caused significant mucosal damage, mast cell infiltration, and goblet cell hyperplasia compared to the control. The transcriptomics results indicated that SABSF significantly enriched multiple inflammatory pathways and showed distinct transcriptional expression differences between the control group and the SABSF collected from CRS patients (p[≤]0.05). Additionally, the SABSF challenges induced the expression of IgA and IgG but not IgE. In conclusion, this in vivo study indicates that long-term exposure to SABSF leads to an inflammatory response in the nasal mucosa with increased severity for S. aureus isolated from a CRSwNP patient. The findings of this study shed light on the role of S. aureus in the development of CRS and could inform future research and treatment efforts.

immunology↗

bioRxiv2023

The Intra-Host Evolutionary Landscape And Pathoadaptation Of Persistent Staphylococcus aureus In Chronic Rhinosinusitis

Chronic rhinosinusitis (CRS) is a common chronic sinonasal mucosal inflammation associated with Staphylococcus aureus biofilm and relapsing infections. This study aimed to determine rates of S. aureus persistence and pathoadaptation in CRS patients by investigating the genomic relatedness and antibiotic resistance/tolerance in longitudinally collected S. aureus clinical isolates. A total of 68 S. aureus isolates were sourced from 34 CRS patients at least six months apart. Isolates were grown into 48-hour biofilms and tested for tolerance to antibiotics. A hybrid sequencing strategy was used to obtain high-quality reference-grade assemblies of all isolates. Single nucleotide variants (SNV) divergence in the core genome and sequence type clustering were used to analyse the relatedness of the isolate pairs. Single nucleotide and structural genome variations, plasmid similarity, and plasmid copy numbers between pairs were examined. Our analysis revealed that 41% (14/34 pairs) of S. aureus isolates were persisters, while 59% (20/34 pairs) were non-persisters. Persister isolates showed episode-specific mutational changes over time with a bias towards events in genes involved in adhesion to the host and mobile genetic elements such as plasmids, prophages, and insertion sequences. A significant increase in the copy number of conserved plasmids of persister strains (p<0.05) was seen, indicating a role of the "mobilome" in promoting persistence. This was accompanied by a significant increase in biofilm tolerance against all tested antibiotics (p<0.001), which was linked to a significant increase in biofilm biomass (p<0.05) over time, indicating a biofilm central pathoadaptive process in persisters. In conclusion, our study provides important insights into the mutational changes underlying S. aureus persistence in CRS patients highlighting pathoadaptive mechanisms in S. aureus persisters culminating in increased biofilm biomass linked to an increase in plasmid copy number over time.

microbiology↗

bioRxiv2023

Novel crAssphage isolates exhibit conserved gene order and purifying selection of the host specificity protein

Bacteroides, the prominent bacteria in the human gut, play a crucial role in degrading complex polysaccharides. Their abundance is influenced by phages belonging to the Crassvirales order. Despite identifying over 600 Crassvirales genomes computationally, only few have been successfully isolated. Continued efforts in isolation of more Crassvirales genomes can provide insights into phage-host-evolution and infection mechanisms. We focused on wastewater samples, as potential sources of phages infecting various Bacteroides hosts. Sequencing, assembly, and characterization of isolated phages revealed 14 complete genomes belonging to three novel Crassvirales species infecting Bacteroides cellulosilyticus WH2. These species, Kehishuvirus sp. tikkala strain Bc01, Kolpuevirus sp. frurule strain Bc03, and Rudgehvirus jaberico strain Bc11, spanned two families, and three genera, displaying a broad range of virion productions. Upon testing all successfully cultured Crassvirales species and their respective bacterial hosts, we discovered that they do not exhibit co-evolutionary patterns with their bacterial hosts. Furthermore, we observed variations in gene similarity, with greater shared similarity observed within genera. However, despite belonging to different genera, the three novel species shared a unique structural gene that encodes the tail spike protein. When investigating the relationship between this gene and host interaction, we discovered evidence of purifying selection, indicating its functional importance. Moreover, our analysis demonstrated that this tail spike protein binds to the TonB-dependent receptors present on the bacterial host surface. Combining these observations, our findings provide insights into phage-host interactions and present three Crassvirales species as an ideal system for controlled infectivity experiments on one of the most dominant members of the human enteric virome. Impact statementBacteriophages play a crucial role in shaping microbial communities within the human gut. Among the most dominant bacteriophages in the human gut microbiome are Crassvirales phages, which infect Bacteroides. Despite being widely distributed, only a few Crassvirales genomes have been isolated, leading to a limited understanding of their biology, ecology, and evolution. This study isolated and characterized three novel Crassvirales genomes belonging to two different families, and three genera, but infecting one bacterial host, Bacteroides cellulosilyticus WH2. Notably, the observation confirmed the phages are not co-evolving with their bacterial hosts, rather have a shared ability to exploit similar features in their bacterial host. Additionally, the identification of a critical viral protein undergoing purifying selection and interacting with the bacterial receptors opens doors to targeted therapies against bacterial infections. Given Bacteroides role in polysaccharide degradation in the human gut, our findings advance our understanding of the phage-host interactions and could have important implications for the development of phage-based therapies. These discoveries may hold implications for improving gut health and metabolism to support overall well-being. Data summaryThe genomes used in this research are available on Sequence Read Archive (SRA) within the project, PRJNA737576. Bacteroides cellulosilyticus WH2, Kehishuvirus sp. tikkala strain Bc01, Kolpuevirus sp. frurule strain Bc03, and Rudgehvirus jaberico strain Bc11 are all available on GenBank with accessions NZ_CP072251.1 (B. cellulosilyticus WH2), QQ198717 (Bc01), QQ198718 (Bc03), and QQ198719 (Bc11), and we are working on making the strains available through ATCC. The 3D protein structures for the three Crassvirales genomes are available to download at doi.org/10.25451/flinders.21946034.

bioinformatics↗