Biofilm-associated Protein Research Articles

Harnessing anti-infective efficacy of Cinnamomum verum in synergy with β-lactam and fluoroquinolones drugs to combat virulence and biofilms of Pseudomonas aeruginosa PAO1

Multidrug resistance (MDR) Gram-negative bacteria are increasingly resistant to multiple antibiotics, posing a serious challenge to infection control and treatment. Combining plant-derived bioactives with antibiotics offers a promising approach to overcome the challenges posed by MDR pathogens like Pseudomonas aeruginosa. This study investigated the synergistic effects of Cinnamomum verum with beta-lactam and fluoroquinolones against P. aeruginosa PAO1. The ethyl acetate fraction of C. verum (CVEF) was obtained through fractionation in organic solvents with progressively higher polarity. The interaction of CVEF with selected antibiotics was assessed by checkerboard synergy assay. The effects of synergistic combinations on pyocyanin, pyoverdine, protease, EPS production, and biofilm development were measured using spectroscopic assays. CVEF combined with cefepime, ceftazidime, and levofloxacin significantly enhanced antibacterial efficacy with FICIs between 0.156 to 0.5. The most active combinations i.e., CVEF-cefepime and CVEF-ceftazidime inhibited viable cell count of growth by 3.6 and 4.2 log10 CFU/ml respectively. The combination also inhibited virulence factors (>75%) and biofilms (>80%) at lower 1/2×FICs. The viable count of biofilm cells was also reduced from 6.4 to 3.3 and 3.6 log10 CFU/ml. Membrane permeability was decreased by 60.34% and biofilm cell viability by 22.53-38.44%. Key phytochemicals analyzed by GC/MS and LC/MS/MS, include cinnamaldehyde, trans-chlorogenic acid, quercetin, and quercetin 3'-O-glucuronide. In molecular docking investigations, quercetin 3'-O-glucuronide had the highest binding affinity with quorum sensing (QS) and biofilm-associated protein. The findings suggest CVEF, in combination with antibiotics, effectively targets resistance phenotypes of P. aeruginosa, impairing growth, virulence, and biofilms. This supports further research into natural compounds alongside antibiotics to treat drug-resistant infections.

Insights into Acinetobacter baumannii AMA205's Unprecedented Antibiotic Resistance.

The rise of antibiotic-resistant bacteria in clinical settings has become a significant global concern. Among these bacteria, Acinetobacter baumannii stands out due to its remarkable ability to acquire resistance genes and persist in hospital environments, leading to some of the most challenging infections. Horizontal gene transfer (HGT) plays a crucial role in the evolution of this pathogen. The A. baumannii AMA205 strain, belonging to sequence type ST79, was isolated from a COVID-19 patient in Argentina in 2021. This strain's antimicrobial resistance profile is notable as it harbors multiple resistance genes, some of which had not been previously described in this species. The AmpC family β-lactamase blaCMY-6, commonly found in Enterobacterales, had never been detected in A. baumannii before. Furthermore, this is the first ST79 strain known to carry the carbapenemase blaNDM-1 gene. Other acquired resistance genes include the carbapenemase blaOXA-23, further complicating treatment. Susceptibility testing revealed high resistance to most antibiotic families, including cefiderocol, with significant contributions from blaCMY-6 and blaNDM-1 genes to the cephalosporin and carbapenem resistance profiles. The A. baumannii AMA205 genome also contains genetic traits coding for 111 potential virulence factors, such as the iron-uptake system and biofilm-associated proteins. This study underscores A. baumannii's ability to acquire multiple resistance genes and highlights the need for alternative therapies and effective antimicrobial stewardship to control the spread of these highly resistant strains.

A membrane localized RTX-like protein mediates physiochemical properties of the Pantoea stewartii subsp. stewartii cell envelope that impact surface adhesion, cell surface hydrophobicity and plant colonization

Pantoea stewartii subsp. stewartii (Pnss), is the bacterial causal agent of Stewart’s wilt of sweet corn. Disease symptoms include systemic wilting and foliar, water-soaked lesions. A Repeat-in-toxin (RTX)-like protein, RTX2, causes cell leakage and collapse in the leaf apoplast of susceptible corn varieties and is a primary mediator of water-soaked lesion formation in the P. stewartii-sweet corn pathosystem. RTX toxins comprise a large family of proteins, which are widely distributed among Gram-negative bacteria. These proteins are generally categorized as cellulolysins, but the Biofilm-Associated Proteins (Bap) subfamily of RTX toxins are implicated in surface adhesion and other biofilm behaviors. The Pnss RTX2 is most phylogenetically related to other Bap proteins suggesting that Pnss RTX2 plays a dual role in adhesion to host surfaces in addition to mediating the host cell lysis that leads to water-soaked lesion formation. Here we demonstrated that RTX2 localizes to the bacterial cell envelope and influences physiochemical properties of the bacterial cell envelope that impact bacterial cell length, cell envelope integrity and overall cellular hydrophobicity. Interestingly, the role of RTX2 as an adhesin was only evident in absence of exopolysaccharide (EPS) production suggesting that RTX2 plays a role as an adhesin early in biofilm development before EPS production is fully induced. However, deletion of rtx2 severely impacted Pnss’ colonization of the xylem suggesting that the dual role of RTX2 as a cytolysin and adhesin is a mechanism that links the apoplastic water-soaked lesion phase of infection to the wilting phase of the infection in the xylem.

BBSdb, an open resource for bacterial biofilm-associated proteins.

Bacterial biofilms are organized heterogeneous assemblages of microbial cells encased within a self-produced matrix of exopolysaccharides, extracellular DNA and proteins. Over the last decade, more and more biofilm-associated proteins have been discovered and investigated. Furthermore, omics techniques such as transcriptomes, proteomes also play important roles in identifying new biofilm-associated genes or proteins. However, those important data have been uploaded separately to various databases, which creates obstacles for biofilm researchers to have a comprehensive access to these data. In this work, we constructed BBSdb, a state-of-the-art open resource of bacterial biofilm-associated protein. It includes 48 different bacteria species, 105 transcriptome datasets, 21 proteome datasets, 1205 experimental samples, 57,823 differentially expressed genes (DEGs), 13,605 differentially expressed proteins (DEPs), 1,930 'Top 5% differentially expressed genes', 444 'Threshold-based DEGs' and a predictor for prediction of biofilm-associated protein. In addition, 1,781 biofilm-associated proteins, including annotation and sequences, were extracted from 942 articles and public databases via text-mining analysis. We used E. coli as an example to represent how to explore potential biofilm-associated proteins in bacteria. We believe that this study will be of broad interest to researchers in field of bacteria, especially biofilms, which are involved in bacterial growth, pathogenicity, and drug resistance. Availability and implementation: The BBSdb is freely available at http://124.222.145.44/#!/.

Exploring the evolutionary and pathogenic role of Acinetobacter baumannii biofilm-associated protein (Bap) through in silico structural modeling.

Acinetobacter species encode for extracellularly secreted Biofilm-associated protein (Bap), a multi-domain protein with variable molecular weights reaching several hundred kilodaltons. Bap is crucial for the development of multi-dimensional structures of mature biofilms. In our investigation, we analyzed 7338 sequences of A. baumannii from the NCBI database and found that Bap or Bap-like protein (BLP) was present in 6422 (87.52%) isolates. Further classification revealed that 12.12% carried Type-1 Bap, 68.44% had Type-2, 6.91% had Type-3, 0.05% had Type-6 or SDF-Type, and 12.51% lacked Bap or BLP. The majority of isolates with Type-1, Type-2, and Type-3 Bap belonged to ST1, ST2, and ST25, respectively. Phylogenetic analysis suggested that Type-1 Bap is the most ancient, while Type-3 and SDF-Type have evolved recently. Studying the interaction of predicted Bap structures with human CEACAM-1 and PIgR showed that Bap with its BIg13 and BIg6 domains interact with the N-terminal domain of CEACAM-1, involving Arg43 and Glu40, involved in CEACAM-1 dimerization. Also, we found that recently evolved Type-3 and SDF-Type Bap showed greater interaction with CEACAM-1 and PIgR. It can be asserted that the evolution of Bap has conferred enhanced virulence characteristics to A. baumannii with increased interaction with CEACAM-1 and PIgR. Using in silico approaches, this study explores the evolutionary, physicochemical, and structural features of A. baumannii Bap and unravels its crucial role in mediating interaction with human CEACAM-1 and PIgR through detailed structure modelling. These findings advance our understanding of A. baumannii Bap and highlight its role in pathogenesis.

Gut microbiota produces biofilm-associated amyloids with potential for neurodegeneration.

Age-related neurodegenerative diseases involving amyloid aggregation remain one of the biggest challenges of modern medicine. Alterations in the gastrointestinal microbiome play an active role in the aetiology of neurological disorders. Here, we dissect the amyloidogenic properties of biofilm-associated proteins (BAPs) of the gut microbiota and their implications for synucleinopathies. We demonstrate that BAPs are naturally assembled as amyloid-like fibrils in insoluble fractions isolated from the human gut microbiota. We show that BAP genes are part of the accessory genomes, revealing microbiome variability. Remarkably, the abundance of certain BAP genes in the gut microbiome is correlated with Parkinson's disease (PD) incidence. Using cultured dopaminergic neurons and Caenorhabditis elegans models, we report that BAP-derived amyloids induce α-synuclein aggregation. Our results show that the chaperone-mediated autophagy is compromised by BAP amyloids. Indeed, inoculation of BAP fibrils into the brains of wild-type mice promote key pathological features of PD. Therefore, our findings establish the use of BAP amyloids as potential targets and biomarkers of α-synucleinopathies.

Insights into the biosynthesis of palladium nanoparticles for oxygen reduction reaction by genetically engineered bacteria of Shewanella oneidensis MR-1.

Owing to the increasing need for green synthesis and environmental protection, the utilization of biological organism-derived carbons as supports for noble-metal electrocatalysts has garnered public interest. Nevertheless, the mechanism by which microorganisms generate nanometals has not been fully understood yet. In the present study, we used genetically engineered bacteria of Shewanella oneidensis MR-1 (∆SO4317, ∆SO4320, ∆SO0618 and ∆SO3745) to explore the effect of surface substances including biofilm-associated protein (bpfA), protein secreted by type I secretion systems (TISS) and type II secretion systems (T2SS), and lipopolysaccharide in microbial synthesis of metal nanoparticles. Results showed Pd/∆SO4317 (the catalyst prepared with the mutant ∆SO4317) shows better performance than other biocatalysts and commercial Pd/C, where the mass activity (MA) and specific activity (SA) of Pd/∆SO4317 are 3.1 and 2.1 times higher than those of commercial Pd/C, reaching 257.49 A g-1 and 6.85 A m-2 respectively. It has been found that the exceptional performance is attributed to the smallest particle size and the presence of abundant functional groups. Additionally, the absence of biofilms has been identified as a crucial factor in the formation of high-quality bio-Pd. Because the absence of biofilm can minimize metal agglomeration, resulting in uniform particle size dispersion. These findings provide valuable mechanical insights into the generation of biogenic metal nanoparticles and show potential industrial and environmental applications, especially in accelerating oxygen reduction reactions.

The prevalence of adhesion and biofilm genes in Staphylococcus aureus isolates from bovine mastitis: A comprehensive meta-analysis.

Mastitis poses significant challenges to the dairy industry, resulting in economic losses and increased veterinary expenses. Staphylococcus aureus is a common cause of bovine mastitis, relying on efficient adhesion and biofilm formation for infection. This study aimed to employ meta-analysis to investigate the occurrence of adhesion and biofilm genes in S. aureus associated with bovine mastitis, as documented in previous studies. This meta-analysis was done according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses, examined 22 eligible articles and revealed varying prevalence rates of adhesion and biofilm genes in S. aureus isolates from bovine mastitis. Among the genes, clfB showed the highest prevalence (p-estimate=0.905), followed by fnbA (p-estimate=0.689) and fnbB (p-estimate=0.502). The icaA and icaD genes also showed a relatively high prevalence (p-estimate=0.694 and 0.814, respectively). Conversely, the biofilm-associated proteins gene had the lowest prevalence (p-estimate=0.043). Subgroup analyses based on mastitis types and publication years revealed no significant differences in gene prevalence. Insufficient data hindered the analysis of fib, sasG , eno and bbp genes. This study provides valuable insights for managing S. aureus-induced bovine mastitis. Additionally, larger-scale research, particularly on less-studied genes, is necessary to comprehend the molecular roles of adhesion and biofilm genes in S. aureus-induced bovine mastitis.

Oral and middle ear delivery of otitis media standard of care antibiotics, but not biofilm-targeted antibodies, alter chinchilla nasopharyngeal and fecal microbiomes

Otitis media (OM) is one of the most globally pervasive pediatric conditions. Translocation of nasopharynx-resident opportunistic pathogens like nontypeable Haemophilus influenzae (NTHi) assimilates into polymicrobial middle ear biofilms, which promote OM pathogenesis and substantially diminish antibiotic efficacy. Oral or tympanostomy tube (TT)-delivered antibiotics remain the standard of care (SOC) despite consequences including secondary infection, dysbiosis, and antimicrobial resistance. Monoclonal antibodies (mAb) against two biofilm-associated structural proteins, NTHi-specific type IV pilus PilA (anti-rsPilA) and protective tip-region epitopes of NTHi integration host factor (anti-tip-chimer), were previously shown to disrupt biofilms and restore antibiotic sensitivity in vitro. However, the additional criterion for clinical relevance includes the absence of consequential microbiome alterations. Here, nine chinchilla cohorts (n = 3/cohort) without disease were established to evaluate whether TT delivery of mAbs disrupted nasopharyngeal or fecal microbiomes relative to SOC-OM antibiotics. Cohort treatments included a 7d regimen of oral amoxicillin-clavulanate (AC) or 2d regimen of TT-delivered mAb, AC, Trimethoprim-sulfamethoxazole (TS), ofloxacin, or saline. Fecal and nasopharyngeal lavage (NPL) samples were collected before and several days post treatment (DPT) for 16S sequencing. While antibiotic-treated cohorts displayed beta-diversity shifts (PERMANOVA, P < 0.05) and reductions in alpha diversity (q < 0.20) relative to baseline, mAb antibodies failed to affect diversity, indicating maintenance of a eubiotic state. Taxonomic and longitudinal analyses showed blooms in opportunistic pathogens (ANCOM) and greater magnitudes of compositional change (P < 0.05) following broad-spectrum antibiotic but not mAb treatments. Collectively, results showed broad-spectrum antibiotics induced significant fecal and nasopharyngeal microbiome disruption regardless of delivery route. Excitingly, biofilm-targeting antibodies had little effect on fecal and nasopharyngeal microbiomes.

Biofilm-associated proteins as potential drug targets

Biofilm-associated proteins as potential drug targets

YajC, a predicted membrane protein, promotes Enterococcus faecium biofilm formation in vitro and in a rat endocarditis model.

Biofilm formation is a critical step in the pathogenesis of difficult-to-treat Gram-positive bacterial infections. We identified that YajC, a conserved membrane protein in bacteria, plays a role in biofilm formation of the clinically relevant Enterococcus faecium strain E1162. Deletion of yajC conferred significantly impaired biofilm formation in vitro and was attenuated in a rat endocarditis model. Mass spectrometry analysis of supernatants of washed ΔyajC cells revealed increased amounts in cytoplasmic and cell-surface-located proteins, including biofilm-associated proteins, suggesting that proteins on the surface of the yajC mutant are only loosely attached. In Streptococcus mutans YajC has been identified in complex with proteins of two cotranslational membrane protein-insertion pathways; the signal recognition particle (SRP)-SecYEG-YajC-YidC1 and the SRP-YajC-YidC2 pathway, but its function is unknown. In S. mutans mutation of yidC1 and yidC2 resulted in impaired protein insertion in the cell membrane and secretion in the supernatant. The E. faecium genome contains all homologous genes encoding for the cotranslational membrane protein-insertion pathways. By combining the studies in S. mutans and E. faecium, we propose that YajC is involved in the stabilization of the SRP-SecYEG-YajC-YidC1 and SRP-YajC-Yid2 pathway or plays a role in retaining proteins for proper docking to the YidC insertases for translocation in and over the membrane.

Comprehensive Genome Analysis of Colistin-Only-Sensitive KPC-2 and NDM1-1-Coproducing Klebsiella pneumoniae ST11 and Acinetobacter baumannii ST2 From a Critically Ill Patient With COVID-19 in Saudi Arabia: Whole Genome Sequencing (WGS) of K. pneumoniae ST11 and A. baumannii ST2.

The COVID-19 pandemic has intensified the issue of multidrug-resistant (MDR) infections, particularly in intensive care units (ICUs). This study documents the first known case of coinfection with two extensively drug-resistant (XDR) bacterial isolates in a critically ill patient with COVID-19 in Saudi Arabia. Both XDR isolates were recovered from blood and were resistant to all tested antimicrobial agents except colistin. Whole genome sequencing (WGS) revealed that the K. pneumoniae isolate KP-JZ107 had sequence type 11 (ST11) and core genome MLST (cgMLST 304742), while the A. baumannii isolate AB-JZ67 had ST2 and cgMLST 785. KP-JZ107 was found to possess the virulence plasmid KpVP-type-1, carbapenemase genes bla NDM and bla KPC , and numerous antimicrobial-resistant genes (ARGs). The AB-JZ67 isolate had several biofilm-related genes, including biofilm-associated protein (BAP), csuE, and pgaB, and multiple ARGs, including bla ADC-25, bla OXA-23, and bla OXA-66. Our findings suggest that the coexistence of KP-JZ107 and AB-JZ67 isolates may indicate their widespread presence in ICUs, requiring comprehensive surveillance studies across all hospitals.

Combating Staphylococcus aureus biofilm formation: the inhibitory potential of tormentic acid and 23-hydroxycorosolic acid.

Plant extracts have been used to treat microbiological diseases for centuries. This study examined plant triterpenoids tormentic acid (TA) and 23-hydroxycorosolic acid (HCA) for their antibiofilm effects on Staphylococcus aureus strains (MTCC-96 and MTCC-7405). Biofilms are bacterial colonies bound by a matrix of polysaccharides, proteins, and DNA, primarily impacting healthcare. As a result, ongoing research is being conducted worldwide to control and prevent biofilm formation. Our research showed that TA and HCA inhibit S. aureus planktonic growth by depolarizing the bacterial membrane. In addition, zone of inhibition studies confirmed their effectiveness, and crystal violet staining and biofilm protein quantification confirmed their ability to prevent biofilm formation. TA and HCA exhibited substantial reductions in biofilm formation for S. aureus (MTCC-96) by 54.85% and 48.6% and for S. aureus (MTCC-7405) by 47.07% and 56.01%, respectively. Exopolysaccharide levels in S. aureus biofilm reduced significantly by TA (25μg/mL) and HCA (20μg/mL). Microscopy, bacterial motility, and protease quantification studies revealed their ability to reduce motility and pathogenicity. Furthermore, TA and HCA treatment reduced the mRNA expression of S. aureus virulence genes. In silico analysis depicted a high binding affinity of triterpenoids for biofilm and quorum-sensing associated proteins in S. aureus, with TA having the strongest affinity for TarO (-7.8kcal/mol) and HCA for AgrA (-7.6kcal/mol). TA and HCA treatment reduced bacterial load in S. aureus-infected peritoneal macrophages and RAW264.7 cells. Our research indicates that TA and HCA can effectively combat S. aureus by inhibiting its growth and suppressing biofilm formation.

Attenuation of quorum sensing regulated virulence functions and biofilm of pathogenic bacteria by medicinal plant Artemisia annua and its phytoconstituent 1, 8-cineole.

The emergence of multidrug resistance (MDR) in bacterial pathogens is a serious public health concern. A significant therapeutic target for MDR infections is the quorum sensing-regulated bacterial pathogenicity. Determining the anti-quorum sensing abilities of certain medicinal plants against bacterial pathogens as well as the in-silico interactions of particular bioactive phytocompounds with QS and biofilm-associated proteins were the objectives of the present study. In this study, 6 medicinal plants were selected based on their ethnopharmacological usage, screened for Anti-QS activity and Artemisia annua leaf extract (AALE) demonstrated pigment inhibitory activity against Chromobacterium violaceum CV12472. Further, the methanol active fraction significantly inhibited the virulence factors (pyocyanin, pyoverdine, rhamnolipid and swarming motility) of Pseudomonas aeruginosa PAO1 and Serratia marcescens MTCC 97 at respective sub-MICs. The inhibition of biofilm was determined using a microtiter plate test and scanning electron microscopy. Biofilm formation was impaired by 70%, 72% and 74% in P. aeruginosa, C. violaceum and S. marcescens, respectively at 0.5xMIC of the extract. The phytochemical content of the extract was studied using GC-MS and 1, 8-cineole was identified as major bioactive compound. Furthermore, 1, 8-cineole was docked with quorum sensing (QS) proteins (LasI, LasR, CviR, and rhlR) and biofilm proteins (PilY1 and PilT). In silico docking and dynamics simulations studies suggested interactions with QS-receptors CviR', LasI, LasR, and biofilm proteins PilY1, PilT for anti-QS activity. Further, 1, 8-cineole demonstrated 66% and 51% reduction in violacein production and biofilm formation, respectively to validate the findings of computational analysis. Findings of the present investigation suggests that 1, 8-cineole plays a crucial role in the QS and biofilm inhibitory activity demonstrated by Artemisia annua extract. RESEARCH HIGHLIGHTS: Artemisia annua leaf extract (AALE) methanol fraction demonstrated broad-spectrum QS and biofilm inhibition Scanning electron microscopy (SEM) confirmed biofilm inhibition Molecular docking and simulation studies suggested positive interactions of 1,8-cineol with QS-receptors and biofilm proteins.

Enhanced immunoprotection against Acinetobacter baumannii infection: Synergistic effects of Bap and BauA in a murine model

BackgroundThe rise of multi-drug resistant Acinetobacter baumannii poses a grave threat to hospital settings, resulting in increased mortality rates and garnering global attention. The formation of biofilms facilitated by biofilm-associated protein (Bap) and the iron absorption capabilities mediated by Baumannii acinetobactin utilization A (BauA) contribute to the persistence and survival of multidrug-resistant strains. In this study, we aimed to investigate the potential of disrupting the function of BauA and Bap simultaneously as a strategy for controlling A. baumannii. MethodsRecombinant Bap and BauA were expressed, purified, and subcutaneously administered individually and in combination to BALB/c mice. Subsequently, mice were intraperitoneally challenged with A. baumannii, and the bacterial load and tissue damage in the spleen, lung, and liver were assessed. Serum samples were evaluated to determine antibody titers in surviving mice. ResultsSpecific IgG antibodies were significantly increased. A combination of the antigens resulted in enhanced titer of specific IgGs in comparison to either BauA or Bap alone. The antibodies remained stable over a seven-month period. The combination of Bap and BauA exhibited superior immunoprotection against A. baumannii infection compared to individual administration, resulting in a further reduction in bacterial load in the liver, spleen, and lungs. The histopathological analysis demonstrated successful protection of the tissues against A. baumannii-induced damage upon administration of the two immunogens. ConclusionsThe combination of Bap and BauA has the potential to target a broader range of A. baumannii strains, including those expressing either Bap or BauA, thereby increasing its efficacy against a diverse array of strains.

Polystyrene microplastics biodegradation by gut bacterial Enterobacter hormaechei from mealworms under anaerobic conditions: Anaerobic oxidation and depolymerization

Synthetic plastic is used throughout daily life and industry, threatening organisms with microplastic pollution. Polystyrene is a major plastic polymer and also widely found sources of plastic wastes and microplastics. Here, we report that Enterobacter hormaechei LG3 (CP118279.1), a facultative anaerobic bacterial strain isolated from the gut of Tenebrio molitor larvae (mealworms) can oxidize and depolymerize polystyrene under anaerobic conditions. LG3 performed biodegradation while forming a biofilm on the plastic surface. PS biodegradation was characterized by analyses of surface oxidation, change in morphology and molecular weights, and production of biodegraded derivative. The biodegradation performance by LG3 was compared with PS biodegradation by Bacillus amyloliquefaciens SCGB1 under both anaerobic and aerobic conditions. In addition, through nanopore sequencing technology, we identified degradative enzymes, including thiol peroxidase (tpx), alkyl hydroperoxide reductase C (ahpC) and bacterioferritin comigratory protein (bcp). Along with the upregulation of degradative enzymes for biodegradation, changes in lipid A and biofilm-associated proteins were also observed after the cells were incubated with polystyrene microplastics. Our results provide evidence for anaerobic biodegradation by polystyrene-degrading bacteria and show alterations in gene expression patterns after polystyrene microplastics treatment in the opportunistic pathogen Enterobacter hormaechei.

Systemic exposure to bacterial amyloid curli alters the gut mucosal immune response and the microbiome, exacerbating Salmonella-induced arthritis

ABSTRACT The Salmonella biofilm-associated amyloid protein, curli, is a dominant instigator of systemic inflammation and autoimmune responses following Salmonella infection. Systemic curli injections or infection of mice with Salmonella Typhimurium induce the major features of reactive arthritis, an autoimmune disorder associated with Salmonella infection in humans. In this study, we investigated the link between inflammation and microbiota in exacerbating autoimmunity. We studied C57BL/6 mice from two sources, Taconic Farms and Jackson Labs. Mice from Taconic Farms have been reported to have higher basal levels of the inflammatory cytokine IL − 17 than do mice from Jackson Labs due to the differences in their microbiota. When we systemically injected mice with purified curli, we observed a significant increase in diversity in the microbiota of Jackson Labs mice but not in that of the Taconic mice. In Jackson Labs, mice, the most striking effect was the expansion of Prevotellaceae. Furthermore, there were increases in the relative abundance of the family Akkermansiaceae and decreases in families Clostridiaceae and Muribaculaceae in Jackson Labs mice. Curli treatment led to significantly aggravated immune responses in the Taconic mice compared to Jackson Labs counterparts. Expression and production of IL − 1β, a cytokine known to promote IL − 17 production, as well as expression of Tnfa increased in the gut mucosa of Taconic mice in the first 24 hours after curli injections, which correlated with significant increases in the number of neutrophils and macrophages in the mesenteric lymph nodes. A significant increase in the expression of Ccl3 in colon and cecum of Taconic mice injected with curli was detected. Taconic mice injected with curli also had elevated levels of inflammation in their knees. Overall, our data suggest that autoimmune responses to bacterial ligands, such as curli, are amplified in individuals with a microbiome that promote inflammation.

Deciphering the antibiofilm potential of 2-Phenylethyl methyl ether (PEME), a bioactive compound of Kewda essential oil against Staphylococcus aureus

Opportunistic pathogenic bacteria and their pathogenicity linked with biofilm infections become a severe issue as they resist the actions of multiple antimicrobial drugs. Naturally derived drugs having antibiofilm properties are more effective than chemically synthesized drugs. The plant derived essential oils are a rich source of phytoconstituents with widespread pharmacological values. In the present investigation, a major phytoconstituent, 2-Phenyl Ethyl Methyl Ether (PEME) of Kewda essential oil extracted from the flowers of Pandanus odorifer was explored for its prospective antimicrobial and anti-biofilm properties against ESKAPE pathogenic bacterial strains, Staphylococcus aureus and MTCC 740. The minimum inhibitory concentration (MIC) of PEME was found to be 50 mM against the tested bacterial strains. A gradual decrease in biofilm production was observed when PEME was treated with the sub-MIC concentration. The reduction in biofilm formation was noticeable from qualitative assay i.e., Congo Red Agar Assay (CRA) and further quantified by crystal violet staining assay. The decline in exopolysaccharides production was quantified, with the highest inhibition against MTCC 740 with a decrease of 71.76 ± 4.56% compared to untreated control. From the microscopic analysis (light and fluorescence microscopic method), PEME exhibited inhibitory effect on biofilm formation on the polystyrene surface. The in silico studies stated that PEME could invariably bind to biofilm associated target proteins. Further, transcriptomic data analysis suggested the role of PEME in the down-regulation of specific genes, agrA, sarA, norA and mepR, which are critically associated with bacterial virulence, biofilm dynamics and drug resistance patterns in S. aureus. Further, qRT-PCR analysis validated the role of PEME on biofilm inhibition by relative downregulation of agrA, sarA, norA and mepR genes. Further, advanced in silico methodologies could be employed in future investigations to validate its candidature as promising anti-biofilm agent.

Formation of a biofilm matrix network shapes polymicrobial interactions.

Staphylococcus aureus colonizes the same ecological niche as many commensals. However, little is known about how such commensals modulate staphylococcal fitness and persistence. Here we report a new mechanism that mediates dynamic interactions between a commensal streptococcus and S. aureus. Commensal Streptococcus parasanguinis significantly increased the staphylococcal biofilm formation in vitro and enhanced its colonization in vivo. A streptococcal biofilm-associated protein BapA1, not fimbriae-associated protein Fap1, is essential for dual-species biofilm formation. On the other side, three staphylococcal virulence determinants responsible for the BapA1-dependent dual-species biofilm formation were identified by screening a staphylococcal transposon mutant library. The corresponding staphylococcal mutants lacked binding to recombinant BapA1 (rBapA1) due to lower amounts of eDNA in their culture supernatants and were defective in biofilm formation with streptococcus. The rBapA1 selectively colocalized with eDNA within the dual-species biofilm and bound to eDNA in vitro, highlighting the contributions of the biofilm matrix formed between streptococcal BapA1 and staphylococcal eDNA to dual-species biofilm formation. These findings have revealed an additional new mechanism through which an interspecies biofilm matrix network mediates polymicrobial interactions.

Acinetobacter baumannii in suspected bacterial infections: Association between multidrug resistance, virulence genes, & biofilm production.

Acinetobacter baumannii has emerged as a nosocomial pathogen with a tendency of high antibiotic resistance and biofilm production. This study aimed to determine the occurrence of A. baumannii from different clinical specimens of suspected bacterial infections and furthermore to see the association of biofilm production with multidrug resistance and expression of virulence factor genes in A. baumannii. A. baumannii was confirmed in clinical specimens by the detection of the blaOXA-51-like gene. Biofilm production was tested by microtitre plate assay and virulence genes were detected by real-time PCR. A. baumannii was isolated from a total of 307 clinical specimens. The isolate which showed the highest number of A. baumannii was an endotracheal tube specimen (44.95%), then sputum (19.54%), followed by pus (17.26%), urine (7.49%) and blood (5.86%), and <2 per cent from body fluids, catheter-tips and urogenital specimens. A resistance rate of 70-81.43 per cent against all antibiotics tested, except colistin and tigecycline, was noted, and 242 (78.82%) isolates were multidrug-resistant (MDR). Biofilm was detected in 205 (66.78%) with a distribution of 54.1 per cent weak, 10.42 per cent medium and 2.28 per cent strong biofilms. 71.07 per cent of MDR isolates produce biofilm (P<0.05). Amongst virulence factor genes, 281 (91.53%) outer membrane protein A (OmpA) and 98 (31.92%) biofilm-associated protein (Bap) were detected. Amongst 100 carbapenem-resistant A. baumannii, the blaOXA-23-like gene was predominant (96%), the blaOXA-58-like gene (6%) and none harboured the blaOXA-24-like gene. The metallo-β-lactamase genes blaIMP-1 (4%) and blaVIM-1(8%) were detected, and 76 per cent showed the insertion sequence ISAba1. The majority of isolates studied were from lower respiratory tract specimens. The high MDR rate and its positive association with biofilm formation indicate the nosocomial distribution of A. baumannii. The biofilm formation and the presence of Bap were not interrelated, indicating that biofilm formation was not regulated by a single factor. The MDR rate and the presence of OmpA and Bap showed a positive association (P<0.05). The isolates co-harbouring different carbapenem resistance genes were the predominant biofilm producers, which will seriously limit the therapeutic options suggesting the need for strict antimicrobial stewardship and molecular surveillance in hospitals.