Swarming Motility Assay Research Articles

Optimized swarming motility assay to identify anti-virulence products against Vibrio parahaemolyticus, a pathogen of farmed shrimp

Swarming motility is a type of movement used by pathogenic flagellated bacteria as virulence factor to colonize surfaces and cause damage to the host. Vibrio parahaemolyticus is a pathogenic flagellated bacterium that increases its virulence by switching from swimmer to swarming cells. The hosts of pathogenic V. parahaemolyticus include farmed shrimp. Therefore, methods to detect and quantify this movement are important to control shrimp diseases caused by pathogenic V. parahaemolyticus strains. We developed an optimized swarming motility assay by identifying the most optimal type of agar, and drying time of the culture medium, agar concentration and volume of the bacterial culture to achieve the fastest swarming motility during the migration of V. parahaemolyticus on Petri dishes during a 24-hour incubation period. The method includes data analysis that could be used as a tool to identify potential anti-virulence products by comparing the slopes of the linearized diameters of the swarming halos of bacteria treated with the products, as they migrate on Petri dishes over a 24-hour incubation period.Here we report:•A simple method for detection and quantification of swarming motility halos of V. parahaemolyticus bacteria.•A method that could be used as a tool to identify potential anti-virulence products.

Characterization and antivibrio activity of chitosan-citral Schiff base calcium complex for a calcium citrate sustained release antibacterial agent

Vibrio parahemolyticus is the “Number one killer” of seafood products. Anti-vibrio agents having low cost and high-safety are urgently needed to supplement the application needs. This work attempted to prepare CS-CT-CCa complex with citral (CT), chitosan (CS) and calcium citrate (CCa) as raw material by microwave-assisted high-pressure homogenization. Additionally the coordination structure and morphology of Bridge-CS-CT-Schiff base/OH-CCa were verified. The prepared CS-CT-CCa had a well-dispersed property (the size: 3.55~9.33 μm and the zeta potential: +38.7~+67.5 mV) and an excellent sustained released ability (sustained release up to 180 min). MIC, Glucose assay, MDA assay, biofilm formation inhibition assay, SEM, swimming and swarming motility assay demonstrated that CS-CT-CCa had strong (MIC of 128 μg/mL) and sustained (more than 12 h) inhibitory effects against V. parahaemolyticus. Meanwhile, CS-CT-CCa could increase the membrane permeability of V. parahaemolyticus and inhibit their biofilm-forming ability in a dose-dependent manner. It could be inferred that the antibacterial activities against V. parahaemolyticus caused inhibition of biofilm formation, swimming and swarming motilities. This study provided necessary data for the further design and development of chitosan antibacterial agents, food and feed additives.

Synergistic Antibacterial Effect of Ethyl Acetate Fraction of Vernonia amygdalina Delile Leaves with Tetracycline against Clinical Isolate Methicillin-Resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa.

Multidrug-resistant bacteria have raised global concern about the inability to fight deadly infectious diseases. Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa are the most common resistant bacteria that are causing hospital infections. The present study was undertaken to investigate the synergistic antibacterial effect of the ethyl acetate fraction of Vernonia amygdalina Delile leaves (EAFVA) with tetracycline against the clinical isolates MRSA and P. aeruginosa. Microdilution was used to establish the minimum inhibitory concentration (MIC). A checkerboard assay was conducted for the interaction effect. Bacteriolysis, staphyloxanthin, and a swarming motility assay were also investigated. EAFVA exhibited antibacterial activity against MRSA and P. aeruginosa with a MIC value of 125 μg/mL. Tetracycline showed antibacterial activity against MRSA and P. aeruginosa with MIC values of 15.62 and 31.25 μg/mL, respectively. The interaction between EAFVA and tetracycline showed a synergistic effect against MRSA and P. aeruginosa with a Fractional Inhibitory Concentration Index (FICI) of 0.375 and 0.31, respectively. The combination of EAFVA and tetracycline induced the alteration of MRSA and P. aeruginosa, leading to cell death. Moreover, EAFVA also inhibited the quorum sensing system in MRSA and P. aeruginosa. The results revealed that EAFVA enhanced the antibacterial activity of tetracycline against MRSA and P. aeruginosa. This extract also regulated the quorum sensing system in the tested bacteria.

Tragopogon oligolepis’ın Fitokimyasal İçeriği, Antioksidan ve Antiquorum Sensing Özelliğinin Belirlenmesi

Objective: In this study it was aimed to examine antiquorum sensing, antioxidant activities by using root and aerial parts extracts of Tragopogon oligolepis. Also phenolic content was detected using HPLC analysis.Material-Method: Antioxidant activity was detected by DPPH, FRAP methods and phenolic content HPLC. Antiquorum sensing activity was investigated by using pyocyanin and swarming motility assay on Pseudomonas aeruginosa PAO1. Results: Phytochemical profile findings showed that 12 components were detected in the root and 10 components in the aerial parts. The main components were found chlorogenic acid and o-coumaric acid. According to the obtained of antioxidant levels the aerial parts extracts of T. oligolepis had the best antioxidant property in our results. The amount of DPPH (0.60 ± 0.01 mg/ml) and phenolic content (6.55±0.18 mg GAE/g sample) was determined to be high in the aerial parts. In the FRAP analysis, high reducing power was found in the roots (12.62±0.36 μmol FeSO4/g sample). According to these results, although T. oligolepis extracts do not reach very high amounts in terms of antioxidant results, it is thought to be a plant that can be evaluated in terms of removing oxidant effects. The results of antiquorum sensing activity showed that both root and aerial parts extract showed strong inhibitory effect on swarming motility 62%, %65 rate respectively.Conclusions: Tragopogon oligolepis, an endemic species, can be evaluated as an antiquorum sensing inhibitor candidate with its phytochemical contents.

PAS Domain-Containing Chemoreceptors Influence the Signal Sensing and Intestinal Colonization of Vibrio cholerae.

Bacterial chemotaxis is the phenomenon in which bacteria migrate toward a more favorable niche in response to chemical cues in the environment. The methyl-accepting chemotaxis proteins (MCPs) are the principal sensory receptors of the bacterial chemotaxis system. Aerotaxis is a special form of chemotaxis in which oxygen serves as the signaling molecule; the process is dependent on the aerotaxis receptors (Aer) containing the Per-Arnt-Sim (PAS) domain. Over 40 MCPs are annotated on the genome of Vibrio cholerae; however, little is known about their functions. We investigated six MCPs containing the PAS domain in V. cholerae El Tor C6706, namely aer2, aer3, aer4, aer5, aer6, and aer7. Deletion analyses of each aer homolog gene indicated that these Aer receptors are involved in aerotaxis, chemotaxis, biofilm formation, and intestinal colonization. Swarming motility assay indicated that the aer2 gene was responsible for sensing the oxygen gradient independent of the other five homologs. When bile salts and mucin were used as chemoattractants, each Aer receptor influenced the chemotaxis differently. Biofilm formation was enhanced by overexpression of the aer6 and aer7 genes. Moreover, deletion of the aer2 gene resulted in better bacterial colonization of the mutant in adult mice; however, virulence gene expression was unaffected. These data suggest distinct roles for different Aer homologs in V. cholerae physiology.

Deletion of the cheZ gene results in the loss of swimming ability and the decrease of adhesion ability to Caco-2 cells in Escherichia coli Nissle 1917.

The aim of this study was to elucidate the biological functions of the motility regulatory protein CheZ in the probiotic strain Escherichia coli Nissle 1917. A cheZ gene deletion strain Nissle 1917ΔcheZ was constructed using the CRISPR/Cas9 two-plasmid system, and the corresponding complemented strain Nissle 1917ΔcheZ/pBR322-cheZ was established. Combined studies of growth kinetics testing, motility assays, swarming motility assays, and bacterial adherence assays were performed to study the motility regulatory protein CheZ-mediated functions in the prototype Nissle 1917 strain, its isogenic cheZ mutant, and the corresponding complemented strain. The growth rate of the cheZ mutant strain was lower than that of the wild-type strain in the exponential growth phase. The motility of the cheZ mutant strain was significantly lower than that of the wild-type strain. And the adhesion ability of ΔcheZ mutant to the Caco-2 cells was significantly lower than that of the wild-type strain and complemented strain. In conclusion, the results presented in our study suggested that the deletion of the cheZ gene in E. coli Nissle 1917 led to a significant reduction of its swimming ability and a subsequent marked decrease of adhesion to the Caco-2 cells.

Assessing Helicobacter pylori motility and biofilm formation in subinhibitory concentrations of antimicrobials

Numerous studies have shown that subinhibitory concentrations of antimicrobials can alter bacterial virulence factors. This study evaluates motility and biofilm formation by H. pylori 43504 grown in subinhibitory concentrations of amoxicillin (AMX), clarithromycin (CLA), or tetracycline (TET). For the swimming and swarming motility assays, H. pylori 43504 suspensions were prepared with the strain alone or with the strain in AMX, CLA, or TET at ½ MIC. Next, the media were incubated at 37 ºC, under microaerophilia. To assess biofilm formation in the presence of one of the antimicrobials at subinhibitory antimicrobial concentrations, bacterial suspensions (109 CFU/mL) were prepared in 2.5% FBS containing AMX, CLA, or TET at ½ MIC. After incubation for 10 days, H. pylori 43504 grown in medium containing AMX, CLA, or TET at ½ MIC presented greater swimming motility and lower swarming motility than the non-treated strain. H. pylori 43504 grown in medium containing AMX, CLA, or TET at ½ MIC showed stronger biofilm production than the non-treated strain. Our results showed that AMX, CLA, or TET at subinhibitory concentrations favors H. pylori 43504 swimming motility and biofilm formation after incubation for 3 days. This may have clinical consequences and make the microorganism difficult to eradicate.

Analysis of Antibiofilm Activities of Bioactive Compounds from Honeyweed (Leonurus sibiricus) Against P. aeruginosa: an In Vitro and In Silico Approach.

Leonurus sibiricus (Red verticilla, honeyweed) is a type of herbaceous plant predominantly found in Asian subcontinents as weed in crop fields and is widely used for treating diabetes, bronchitis, and menstrual irregularities. However, there is a dearth of study in the application of the plant phytocompounds for treating biofilm-associated chronic infections. The bioactive compounds mainly comprise of tri-terpenes, di-terpenes, phenolic acid, and flavonoids which may have potential role as antimicrobial and antibiofilm agents. Acute and chronic infection causing microbes usually form biofilm and develop virulence factors and antibiotic resistance through quorum sensing (QS). In this study, the bioactive compounds leosibirin, sibiricinone A, leosibirone A, leonotin, quercetin, lavandulifolioside, and myricetin were identified using GC-MS analysis. These were used for analyzing the antibiofilm and anti-quorum sensing activities (rhamnolipid, AHL assay, swarming motility assay) against the biofilm formed by Pseudomonas aeruginosa, the most significant nosocomial disease-causing bacteria. The compounds were able to bring about maximum inhibition in biofilm formation and QS. Although the antibiofilm activity of the phytoextract was found to be higher than that of individual phytocompounds at a concentration of 250µg/mL, quercetin and myricetin showed highest antibiofilm activity against Pseudomonas aeruginosa, respectively, at MIC values of 135µg/mL and 150µg/mL against P aeruginosa. FT-IR study also revealed that the active ingredients were able to bring about the destruction of exopolysaccharides (EPS). These observations were further validated by molecular docking interactions that showed the active ingredients inhibit the functioning of QS sensing proteins by binding with them. It was observed that myricetin showed better interactions with the QS proteins of P. aeruginosa. Myricetin and quercetin show considerable inhibition of biofilm in comparison to the phytocompounds. Thus, the present study suggests that the active compounds from L. sibiricus can be used as an alternate strategy in inhibiting the biofilm formed by pathogenic organisms.

Inhibitory Effects of Artificial Sweeteners on Bacterial Quorum Sensing.

Despite having been tagged as safe and beneficial, recent evidence remains inconclusive regarding the status of artificial sweeteners and their putative effects on gut microbiota. Gut microorganisms are essential for the normal metabolic functions of their host. These microorganisms communicate within their community and regulate group behaviors via a molecular system termed quorum sensing (QS). In the present study, we aimed to study the effects of artificial sweeteners on this bacterial communication system. Using biosensor assays, biophysical protein characterization methods, microscale thermophoresis, swarming motility assays, growth assays, as well as molecular docking, we show that aspartame, sucralose, and saccharin have significant inhibitory actions on the Gram-negative bacteria N-acyl homoserine lactone-based (AHL) communication system. Our studies indicate that these three artificial sweeteners are not bactericidal. Protein-ligand docking and interaction profiling, using LasR as a representative participating receptor for AHL, suggest that the artificial sweeteners bind to the ligand-binding pocket of the protein, possibly interfering with the proper housing of the native ligand and thus impeding protein folding. Our findings suggest that these artificial sweeteners may affect the balance of the gut microbial community via QS-inhibition. We, therefore, infer an effect of these artificial sweeteners on numerous molecular events that are at the core of intestinal microbial function, and by extension on the host metabolism.

Metagenomic Insights Into the Structure and Function of Intestinal Microbiota of the Hadal Amphipods.

Hadal trenches are the deepest known areas of the ocean. Amphipods are considered to be the dominant scavengers in the hadal food webs. The studies on the structure and function of the hadal intestinal microbiotas are largely lacking. Here, the intestinal microbiotas of three hadal amphipods, Hirondellea gigas, Scopelocheirus schellenbergi, and Alicella gigantea, from Mariana Trench, Marceau Trench, and New Britain Trench, respectively, were investigated. The taxonomic analysis identified 358 microbial genera commonly shared within the three amphipods. Different amphipod species possessed their own characteristic dominant microbial component, Psychromonas in H. gigas and Candidatus Hepatoplasma in A. gigantea and S. schellenbergi. Functional composition analysis showed that “Carbohydrate Metabolism,” “Lipid Metabolism,” “Cell Motility,” “Replication and Repair,” and “Membrane Transport” were among the most represented Gene Ontology (GO) Categories in the gut microbiotas. To test the possible functions of “Bacterial Chemotaxis” within the “Cell Motility” category, the methyl-accepting chemotaxis protein (MCP) gene involved in the “Bacterial Chemotaxis” pathway was obtained and used for swarming motility assays. Results showed that bacteria transformed with the gut bacterial MCP gene showed significantly faster growths compared with the control group, suggesting MCP promoted the bacterial swimming capability and nutrient utilization ability. This result suggested that hadal gut microbes could promote their survival in poor nutrient conditions by enhancing chemotaxis and motility. In addition, large quantities of probiotic genera were detected in the hadal amphipod gut microbiotas, which indicated that those probiotics would be possible contributors for promoting the host’s growth and development, which could facilitate adaptation of hadal amphipods to the extreme environment.

Evaluation of Anti-quorum Sensing Potential of Saraca asoca (Family Caesalpiniaceae) against Chromobacterium violaceum and Pseudomonas aeruginosa PA01

Aim: The present study was performed to evaluate the anti-quorum sensing (QS) potential of traditional medicinal herb Saracaasoca (family Caesalpiniaceae) stem bark extract against Chromobacterium violaceum and Pseudomonas aeruginosa PA01.
 Study Design: First, the test sample (bark extract) was screened for anti-QS activity. Then systematic in-vitro and biochemical tests were performed to evaluate the effect of the test sample on the QS mediated virulence factors.
 Place and Duration of Study: All the experimental works were performed in Lab 311, pharmacology division, CSIR-NBRI Lucknow from June 2019 to October 2019.
 Methodology: The samples of Saraca asoca stem bark were washed, dried and extracted using 70% methanol. The minimum inhibitory concentration (MIC) of the prepared Sarca asoca bark extract was determined using the Alamar blue assay, and the anti-QS activity was screened using standard agar overlay method against CV 12472 at subinhibitory concentrations 100, 200 and 300 µg (< MIC value). SAE effect on biofilms formation was assessed by growing biofilms on glass slides in a static culture of PA01. Anti-virulence effect of SAE on the production of QS-regulated virulence factors such as Pyocyanin, proteases, elastases, rhamnolipid and alginate in Pseudomonas aeruginosa was determined using the supernatant of a 24 hours old broth culture of PA01 supplemented with SAE. Using the agar plate technique, the swimming and swarming motility assays were conducted on 0.3% and 0.5% agar plates respectively. One-way ANOVA was used to analyze the data, presented as mean ± SD (standard deviation) of three independent experiments.
 Results: Preliminary screening results showed significant QS inhibition against CV 12472 in an agar overlay disk diffusion assay in a concentration-dependent manner. Data from the biofilm assay showed loose, distorted, irregular PA01 biofilm formation at 200 µg (48%) and 300 µg (65%). SAE caused a significant drop in virulence factor production, with maximum reduction in pyocyanin (58%), proteases (67%), elastases (52%), rhamnolipid (53%), and alginate (44%) observed at 300 µg concentration. At SAE sub-lethal concentrations (200 and 300 µg), both the swimming and swarming motility of PA01 were significantly inhibited.
 Conclusions: The present study demonstrates the broad-spectrum anti-QS potential of SAE, reported for the first time, suggesting that SAE could be considered as an alternative herbal source to develop antimicrobial agents which can be either solitary or synergistically with conventional antimicrobial drugs.

In Vitro Quorum Quenching Activity of Eleusine indica Crude Ethanolic Extract against Pseudomonas aeruginosa and Serratia marcescens

Introduction. Nosocomial contaminants such as Pseudomonas aeruginosa and Serratia marcescens are increasingly developing resistance to many antibiotics. One of the promising alternatives that may complement, if not substitute, the use of antibiotics is quorum quenching, the process of interfering with chemical signals that mediate communication between microorganisms. Eleusine indica, a ubiquitous grass used traditionally to treat infections, has been shown to contain metabolites, such as fatty acid derivatives and p-coumaric acid, capable of quorum quenching. To date, there has been no study on the quorum quenching activity of E. indica. Objectives. This study aimed to determine the in vitro activity of crude ethanolic extract of E. indica leaves against selected quorum-sensing regulated virulence factors of P. aeruginosa and S. marcescens. Methodology. E. indica leaves were collected, washed, air-dried, and homogenized. Following ethanolic extraction and rotary evaporation, the extract was screened for antimicrobial activity through disk diffusion test and broth microdilution assay. The quorum quenching activity of the extract against P. aeruginosa was measured through swarming motility assay, while the activity against S. marcescens was measured through swarming motility and pigment inhibition assays. The quorum quenching assays were conducted in triplicates, and analysis of variance (ANOVA) was performed to identify differences among the treatment groups. Results. Disk diffusion test revealed that no zones of inhibition formed against both P. aeruginosa and S. marcescens for varying concentrations of up to 200 mg/mL of the crude extract. Likewise, the MIC of the extract against both P. aeruginosa and S. marcescens was determined to be >200 mg/mL. However, it was shown that the extract, at 50 mg/mL, has statistically significant activity (p<0.05) against the swarming motility of P. aeruginosa, and it is 71.6% as effective in reducing the swarming area of the bacteria compared to cinnamaldehyde. This was not observed when the extract was tested against the swarming motility of and pigment production by S. marcescens. Conclusion. In this study, the quorum quenching activity of the crude ethanolic extract of E. indica leaves was found to be effective against P. aeruginosa but not against S. marcescens. The compounds that will be identified by further studies may conceivably be used as an adjunct therapy in P. aeruginosa infections and as coating agents in medical devices.

Design, synthesis, and antitumor evaluation of novel methylene moiety-tetheredtetrahydroquinoline derivatives

Novel methylene-tethered tetrahydroquinolines (THQs) and cyclopenta[$b$]pyridines were synthesized by one-pot multicomponent reactions of Mannich bases, enolizable ketones, and NH$_{4}$OAc in water by an environmentally friendly K-10 montmorillonite clay-catalyzed reaction. The cytotoxic activities of 1-(2-methyl-8-methylene-5,6,7,8-tetrahydroquinolin-3-yl)ethanone (9a), ethyl 2-methyl-8-methylene-5,6,7,8-tetrahydroquinoline-3 carboxylate (9b), and 1-(2-methyl-7-methylene-6,7-dihydro-5$H$-cyclopenta[$b$]pyridin-3-yl)ethanone (11a) were tested against rat glioblastoma (C6), human breast cancer (MCF-7), prostate cancer (PC3), neuroblastoma (SH-SY5Y), and mouse fibroblast (L929) cell lines in a concentration-dependent (50-300 μM) and time-dependent (24-72 h) manner and expressed as IC$_{50\, }$values. The results showed that compound 9a induced the lowest IC$_{50\, }$values in all cell lines ranging from 111 p 1.1 μM to 128 p 1.3 μM when compared to 9b and 11a after 72 h. As an evaluation of antibacterial properties, a swarming motility assay was performed with the Pseudomonas aeruginosa PA01 strain and compound 9a showed higher inhibition of swarming motility.

Quorum sensing regulates the transcription of lateral flagellar genes in Vibrio parahaemolyticus.

Aim: Investigation of the lateral flagellar (Laf) genes transcription by the quorum sensing (QS) regulators AphA and OpaR in Vibrio parahaemolyticus. Materials & methods: Regulation mechanisms were assessed by combined utilization of swarming motility assay, qPCR, LacZ fusion, EMSA and DNase I footprinting. Results: AphA and OpaR oppositely regulate swarming motility and Laf genes. At high cell density, OpaR bound to the regulatory regions of motY-lafK-fliEFGHIJ, fliMNPQR-flhBA, fliDSTKLA-motAB and lafA to repress their transcription. At low cell density, AphA indirectly activated their transcription. Conclusion: OpaR repression of swarming motility was via its direct repression of Laf genes, while AphA exerted its regulatory effect on swarming motility through unknown regulator(s).

Chitosan and graphene oxide hybrid nanocomposite film doped with silver nanoparticles efficiently prevents biofouling

We developed four different hybrid nanocomposite films using graphene oxide nanosheet (GOns), chitosan (Ch) and silver nanoparticles (AgNP) and investigated their inhibitory effect on bacterial biofilm formation (biofouling). Spectroscopy and electron microscopy results confirmed the doping of AgNP on GOns-Ch matrix. The wettability study of films also confirmed that GOns-Ch-AgNP nanocomposite film has the highest surface hydrophobicity among all. When Escherichia coli and Bacillus subtilis were grown and biofouling was studied with different nanocomposite films, AgNP-doped nanocomposite film demonstrated efficient inhibition of biofilm formation which was evident by agar plate, congo red, swarming motility assay as well as electron microscopy imaging results. We also observed from the degradation assay that the nanocomposites were pretty stable in solution and showed ignorable leaching of AgNP and minimum structural deformation of the matrix. Overall, our study clearly proved that AgNP doped GOns-Ch nano-composite (GOns-Ch-AgNP) exhibited anti-adhesion as well as anti-microbial property and hence the nanocomposite might be used as a surface coating material in various industries including marine and health sector industries to prevent biofouling.

Influence of twitching and swarming motilities on biofilm formation in Pseudomonas strains.

The genus Pseudomonas mainly includes opportunistic pathogens that rely on type IV pili as an important virulence factor, which is associated with adherence and biofilm formation. Pseudomonas infections are well known to be persistent and resilient in nature largely because of the tendency of the species to form biofilms. This study aimed at analyzing environmental strains of Pseudomonas genus with respect to their ability to execute twitching and swarming motilities as well as with respect to their ability to form biofilms both in the presence as well as in the absence of furanone, a substance that has the potential to prevent the formation of biofilms. Strains of Pseudomonas aeruginosa and strains belonging to other species of the genus were analyzed. Twitching and swarming motility assays and biofilm-formation assays, both in the presence as well as in the absence of furanone, were performed. In twitching assay strains belonging to P. aeruginosa outperformed those belonging to other species. Interestingly, it was seen that the presence of furanone had a negative impact on formation of twitching and swarming motility zones. In the case of biofilm assays, it was observed that the presence of furanone resulted in an observable decrease in the degree of adhesion in 30% of the analyzed strains. Thus, from our results, it can be concluded that, as compared to other species, the strains belonging to P. aeruginosa exhibit a higher potential for twitching motility and similar performance in swarming motility and biofilm formation. It can also be concluded that furanone has the potential to interfere with both motilities as well as with biofilm formation.

Characterization of Novel Factors Involved in Swimming and Swarming Motility in Salmonella enterica Serovar Typhimurium.

Salmonella enterica utilizes flagellar motility to swim through liquid environments and on surfaces. The biosynthesis of the flagellum is regulated on various levels, including transcriptional and posttranscriptional mechanisms. Here, we investigated the motility phenotype of 24 selected single gene deletions that were previously described to display swimming and swarming motility effects. Mutations in flgE, fliH, ydiV, rfaG, yjcC, STM1267 and STM3363 showed an altered motility phenotype. Deletions of flgE and fliH displayed a non-motile phenotype in both swimming and swarming motility assays as expected. The deletions of STM1267, STM3363, ydiV, rfaG and yjcC were further analyzed in detail for flagellar and fimbrial gene expression and filament formation. A ΔydiV mutant showed increased swimming motility, but a decrease in swarming motility, which coincided with derepression of curli fimbriae. A deletion of yjcC, encoding for an EAL domain-containing protein, increased swimming motility independent on flagellar gene expression. A ΔSTM1267 mutant displayed a hypermotile phenotype on swarm agar plates and was found to have increased numbers of flagella. In contrast, a knockout of STM3363 did also display an increase in swarming motility, but did not alter flagella numbers. Finally, a deletion of the LPS biosynthesis-related protein RfaG reduced swimming and swarming motility, associated with a decrease in transcription from flagellar class II and class III promoters and a lack of flagellar filaments.

Anti-biofilm activity of Marula – A study with the standardized bark extract

Ethnopharmacological relevanceMarula (Sclerocarya birrea; family – Anacardiaceae) is an African plant, which enjoys wide socio-economic importance particularly in southern part of Africa. The fruits are consumed as food and also as alcoholic beverage (cream liquor). In different parts of Africa, the decoction of the bark is traditionally used for the treatment of dysentery, diarrhoea, and various other infectious conditions. The aim of the study was to investigate the anti-biofilm properties of the methanol extract of Marula bark (stem bark of Sclerocarya birrea), with a view towards combating the emergence of antimicrobial resistance often associated with bacterial biofilms. Materials and methodsThe standardized methanol extract was initially tested for its antimicrobial property. The crystal violet assay was used for evaluating anti-biofilm (biofilm formation by Pseudomonas aeuginosa) activity. Further in order to study the mechanism of anti-biofilm activity, the same was evaluated for understanding its role on various quorums sensing mediated phenomenon (swarming motility assay, protease and pyoverdin assay) that are known to be associated with the formation of biofilms and pathogenicity. ResultsThe methanol extract showed no inhibition of bacterial growth up to a concentration of 200µg/ml. Interestingly, the sample produced anti-biofilm activity (around 75% decrease; 100µg/ml) at sub-lethal concentration. Further it also significantly reduced the QS mediated swarming motility. The release of various virulent factors (protease and pyoverdin) was found to be lowered when pre-treated with the extract. ConclusionThe present study illustrates the anti-biofilm property Sclerocarya birrea. The standardized extract significantly disrupted the quorum sensing mediated production of biofilm formation and also inhibited swarming ability of the cells. The extract displayed a regulatory role on the secretion of protease and pyoverdin, two QS dependent pathogenic factors found in Pseudomonas aeruginosa. This study also validates the ethnobotanical use of Marula.

Plate-based assay for swarming motility in Pseudomonas aeruginosa.

Swarming motility is one of three distinct modes of motility observed in the gram-negative bacterium Pseudomonas aeruginosa. Swarming motility is defined as the movement across a semisolid surface, and in P. aeruginosa requires flagellar motility and the production of biosurfactants. Swarming motility is thought to occur on gelatinous/viscous surfaces inside a host, such as on epithelial cells. There is currently no standardized in vitro assay to visualize and study swarming motility, and the assays used can vary greatly between laboratory groups. Here, we describe a detailed, reproducible in vitro swarming motility assay for P. aeruginosa. While different protocols have previously been reported in the literature, we hope that adopting this method will improve the reproducibility of these swarming motility assays and allow comparisons of swarming motility findings between and among groups.

Evaluation of Anti-Quorum Sensing Activity of 97 Indigenous Plant Extracts From Korea through Bioreporter Bacterial Strains Chromobacterium violaceum and Pseudomonas aeruginosa

Quorum sensing (QS) is a recently discovered chemical communication system that enhances survival of bacteria, as a group allowing resident bacteria to assume specialized roles vital for intra- and inter bacterial gene regulation, and for keeping bacterial colonies intact. Furthermore, with the continuing emergence and spread of multidrug-resistant bacteria, antipathogenic strategy to combat bacterial infections through the interruption of quorum sensing controlled virulence factors had been shown to receive increased attention. With this prospect in the current study, we attempt to screen anti-quorum sensing activity of 97 indigenous plant extracts from Korea, through biomonitor bacterial strains, Chromobacterium violaceum (CV12472) and Pseudomonas aeruginosa (PAO1). Standard disc-diffusion assays were used to detect anti-quorum sensing activity (ring of colorless, but viable cells around the disk), of the plant extracts for CV12472. A special swarm media that allow swarming motility growth of POA1 was used to conduct inhibition of swarming motility assay. Minimum Inhibitory Concentration (MIC) test for the 97 plant extracts against bioreporter strains (CV12472 and PAO1) revealed antibacterial activity of three plant extracts (Potentilla cryptotaeniae, Viburnum carlesii and Prunus armeniaca var. ansu). Out of the 97 plant extracts, significant inhibition of pigment production were detected by six plant extracts in CV12472, while 16 plant extracts had shown inhibition of swarming motility in POA1. In conclusion, a total of 18 plant extracts were screened for their anti-quorum sensing activity by the two bioreporter strains. Of the 18 plant extracts, four had shown anti-quorum activities in both bioreporter strains.