Swimming Motility Research Articles

Effects of Sub-Minimum Inhibitory Concentrations of Bacteriocin BM173 on Listeria Monocytogenes Biofilm Formation.

Listeria monocytogenes is a significant foodborne pathogen that can form biofilms on various food processing surfaces, thereby enhancing resistance to disinfectants and exacerbating harm to human health. Previous studies have indicated that bacteriocin BM173 exhibits antibacterial and antibiofilm activities. In the current study, our aim was to assess the inhibitory mode of action of sub-inhibitory concentrations (SICs, 1/32 × MIC and 1/16 × MIC) of BM173 on the biofilm formation L. monocytogenes. Crystal violet staining assay revealed that SICs of BM173 significantly inhibit L. monocytogenes biofilm formation. Furthermore, the results of swimming motility assay, plate count, ruthenium red staining, and scanning electron microscopy (SEM) revealed that SICs of BM173 could effectively reduce the movement, cell adhesion, and exopolysaccharide (EPS) production of L. monocytogenes, thereby inhibiting biofilm formation. Real-time quantitative PCR analyses further demonstrated that SICs of BM173 down-regulated the expression of biofilm-associated genes, including those encoding adhesion, virulence factors, and quorum sensing. Additionally, SICs of BM173 effectively reduced the biofilm formation of L. monocytogenes on the surfaces of three food-grade materials (glass, stainless steel, and silicone) at 4 and 25 °C. These outcomes suggest that BM173 holds great potential for development as a promising food preservative for application in the food industry.

Molecular basis of the phenotypic variants arising from a Pseudoalteromonas lipolytica mutator.

Bacterial deficiencies in the DNA repair system can produce mutator strains that promote adaptive microevolution. However, the role of mutator strains in marine Pseudoalteromonas, capable of generating various gain-of-function genetic variants within biofilms, remains largely unknown. In this study, inactivation of mutS in Pseudoalteromonas lipolytica conferred an approximately 100-fold increased resistance to various antibiotics, including ciprofloxacin, rifampicin and aminoglycoside. Furthermore, the mutator of P. lipolytica generated variants that displayed enhanced biofilm formation but reduced swimming motility, indicating a high phenotypic diversity within the ΔmutS population. Additionally, we observed a significant production rate of approximately 50 % for the translucent variants, which play important roles in biofilm formation, when the ΔmutS strain was cultured on agar plates or under shaking conditions. Using whole-genome deep-sequencing combined with genetic manipulation, we demonstrated that point mutations in AT00_17115 within the capsular biosynthesis cluster were responsible for the generation of translucent variants in the ΔmutS subpopulation, while mutations in flagellar genes fliI and flgP led to a decrease in swimming motility. Collectively, this study reveals a specific mutator-driven evolution in P. lipolytica, characterized by substantial genetic and phenotypic diversification, thereby offering a reservoir of genetic attributes associated with microbial fitness.

Mutational hotspots lead to robust but suboptimal adaptive outcomes in certain environments.

The observed mutational spectrum of adaptive outcomes can be constrained by many factors. For example, mutational biases can narrow the observed spectrum by increasing the rate of mutation at isolated sites in the genome. In contrast, complex environments can shift the observed spectrum by defining fitness consequences of mutational routes. We investigate the impact of different nutrient environments on the evolution of motility in Pseudomonas fluorescens Pf0-2x (an engineered non-motile derivative of Pf0-1) in the presence and absence of a strong mutational hotspot. Previous work has shown that this mutational hotspot can be built and broken via six silent mutations, which provide rapid access to a mutation that rescues swimming motility and confers the strongest swimming phenotype in specific environments. Here, we evolved a hotspot and non-hotspot variant strain of Pf0-2x for motility under nutrient-rich (LB) and nutrient-limiting (M9) environmental conditions. We observed the hotspot strain consistently evolved faster across all environmental conditions and its mutational spectrum was robust to environmental differences. However, the non-hotspot strain had a distinct mutational spectrum that changed depending on the nutrient environment. Interestingly, while alternative adaptive mutations in nutrient-rich environments were equal to, or less effective than, the hotspot mutation, the majority of these mutations in nutrient-limited conditions produced superior swimmers. Our competition experiments mirrored these findings, underscoring the role of environment in defining both the mutational spectrum and the associated phenotype strength. This indicates that while mutational hotspots working in concert with natural selection can speed up access to robust adaptive mutations (which can provide a competitive advantage in evolving populations), they can limit exploration of the mutational landscape, restricting access to potentially stronger phenotypes in specific environments.

Phase variation modulates the multi-phenotypes displayed by clinical Campylobacter jejuni strains

The high incidence and prevalence of Campylobacter spp. in the food supply chain entail the importance to understand their mechanisms developed to withstand harsh environmental conditions encountered. Different stress conditions and phenotypic approaches were evaluated to study the behaviour of five clinical C. jejuni isolates with different genotypes, including the tolerance to oxygen and the oxidants hydrogen peroxide and cumene hydroperoxide, the motility and the ability to form biofilm on polystyrene and stainless steel at different temperatures and atmospheres. Whole Genome Sequencing was performed to analyse the occurrence of 216 genes involved in these mechanisms plus phase variation. The isolates showed high tolerance to oxygen and peroxide stress with different swimming motility performances and biofilm formation abilities. Aerotolerance was related with a reduced sensitive to peroxide stress and a loss of motility that promotes biofilm formation depending on the material surface. Comparative genomics did not reveal any clear gene pattern, although phase variation occurring during host infection was observed to be crucial for the modulation of the different survival mechanisms adopted by the bacteria. These findings reveal that the bacteria can combine diverse and complex strategies in an efficient manner to survive and persist in the environment.

PlzD modifies Vibrio vulnificus foraging behavior and virulence in response to elevated c-di-GMP.

Many free-swimming bacteria propel themselves through liquid using rotary flagella, and mounting evidence suggests that the inhibition of flagellar rotation initiates biofilm formation, a sessile lifestyle that is a nearly universal surface colonization paradigm in bacteria. In general, motility and biofilm formation are inversely regulated by the intracellular second messenger bis-(3´-5´)-cyclic dimeric guanosine monophosphate (c-di-GMP). Here, we identify a protein, PlzD, bearing a conserved c-di-GMP binding PilZ domain that localizes to the flagellar pole in a c-di-GMP-dependent manner and alters the foraging behavior, biofilm, and virulence characteristics of the opportunistic human pathogen, Vibrio vulnificus. Our data suggest that PlzD interacts with components of the flagellar stator to decrease bacterial swimming speed and changes in swimming direction, and these activities are enhanced when cellular c-di-GMP levels are elevated. These results reveal a physical link between a second messenger (c-di-GMP) and an effector (PlzD) that promotes transition from a motile to a sessile state in V. vulnificus.

Alkaline phosphatase LapA regulates quorum sensing-mediated virulence and biofilm formation in Pseudomonas aeruginosa PAO1 under phosphate depletion stress.

Among various environmental stresses, the depletion of inorganic phosphate, which is an important signaling molecule that modulates virulence production, is notable. In our previous study, we demonstrated that lapA, encoding alkaline phosphatase, plays a key role in Pseudomonas aeruginosa biofilm formation in porcine skin explants, an ex vivo chronic wound model. However, the mechanism by which lapA participates in the virulence and biofilm formation of P. aeruginosa remains unclear. In the present study, the lapA deletion mutant was constructed in P. aeruginosa PAO1, and the phenotypic characterization was assessed. Our data demonstrated that phosphate depletion stress significantly enhanced elastase activity, hemolysis, rhamnolipid production, swarming and swimming motilities, and 3-oxo-C12-homoserine lactone (HSL) production in P. aeruginosa. Moreover, phosphate starvation evidently induced the virulence of P. aeruginosa in Caenorhabditis elegans (C. elegans) through fast-kill and slow-kill infections. Deletion of the lapA gene led to broad phenotypic changes, including reduced elastase activity, swimming motility, C4-HSL, and 3-oxo-C12-HSL production, as well as increased chitinase activity and rhamnolipid production under phosphate depletion stress, which was unrelated to C4-HSL. Deletion of the lapA gene also significantly inhibited PAO1 biofilm formation in porcine skin explants by reducing the expression levels of las and rhl quorum sensing systems and extracellular polymeric substance synthesis. Finally, lapA deletion also reduced the virulence of PAO1 in C. elegans in fast-kill and slow-kill infection assays. Thus, the findings suggest that targeting lapA can help control biofilm formation and reduce virulence in nonhealing-infected wounds, where P. aeruginosa is a persistent problem. IMPORTANCE Our previous study demonstrated that the expression of lapA was induced under phosphate depletion conditions, but its roles in virulence and biofilm formation by Pseudomonas aeruginosa remain largely unknown. This study presents a systematic investigation of the roles of lapA in virulence induction and biofilm formation by constructing a lapA-deficient strain with P. aeruginosa PAO1. The results showed that deletion of the lapA gene evidently reduced elastase activity, swimming motility, C4-HSL, and 3-oxo-C12-HSL production, and increased rhamnolipid production under phosphate depletion stress. Moreover, lapA gene deletion inhibited PAO1 biofilm formation in porcine skin explants by reducing the expression levels of las and rhl quorum sensing systems and extracellular polymeric substance synthesis. Finally, lapA gene deletion also reduced the virulence of PAO1 in Caenorhabditis elegans in fast-kill and slow-kill infection assays. This study provides insights into the roles of lapA in modulating P. aeruginosa virulence and biofilm formation under phosphate depletion stress.

The multidrug efflux pump regulator AcrR directly represses motility in Escherichia coli.

Efflux and motility are two key biological functions in bacteria. Recent findings have shown that efflux impacts flagellum biosynthesis and motility in Escherichia coli and other bacteria. AcrR is known to be the major transcriptional repressor of AcrAB-TolC, the main multidrug efflux pump in E. coli and other Enterobacteriaceae. However, the underlying molecular mechanisms of how efflux and motility are co-regulated remain poorly understood. Here, we have studied the role of AcrR in direct regulation of motility in E. coli. By combining bioinformatics, electrophoretic mobility shift assays (EMSAs), gene expression, and motility experiments, we have found that AcrR represses motility in E. coli by directly repressing transcription of the flhDC operon, but not the other flagellum genes/operons tested. flhDC encodes the master regulator of flagellum biosynthesis and motility genes. We found that such regulation primarily occurs by direct binding of AcrR to the flhDC promoter region containing the first of the two predicted AcrR-binding sites identified in this promoter. This is the first report of direct regulation by AcrR of genes unrelated to efflux or detoxification. Moreover, we report that overexpression of AcrR restores to parental levels the increased swimming motility previously observed in E. coli strains without a functional AcrAB-TolC pump, and that such effect by AcrR is prevented by the AcrR ligand and AcrAB-TolC substrate ethidium bromide. Based on these and prior findings, we provide a novel model in which AcrR senses efflux and then co-regulates efflux and motility in E. coli to maintain homeostasis and escape hazards. IMPORTANCE Efflux and motility play a major role in bacterial growth, colonization, and survival. In Escherichia coli, the transcriptional repressor AcrR is known to directly repress efflux and was later found to also repress flagellum biosynthesis and motility by Kim et al. (J Microbiol Biotechnol 26:1824-1828, 2016, doi: 10.4014/jmb.1607.07058). However, it remained unknown whether AcrR represses flagellum biosynthesis and motility directly and through which target genes, or indirectly because of altering the amount of efflux. This study reveals that AcrR represses flagellum biosynthesis and motility by directly repressing the expression of the flhDC master regulator of flagellum biosynthesis and motility genes, but not the other flagellum genes tested. We also show that the antimicrobial, efflux pump substrate, and AcrR ligand ethidium bromide regulates motility via AcrR. Overall, these findings support a novel model of direct co-regulation of efflux and motility mediated by AcrR in response to stress in E. coli.

Anti-Biofilm Activity of Laurel Essential Oil against Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a primary seafood-associated pathogen that could cause gastroenteritis. It can attach to various surfaces and form a biofilm, which poses serious threats to food safety. Hence, an effective strategy is urgently needed to control the biofilm formation of V. parahaemolyticus. Laurel essential oil (LEO) is used in food, pharmaceutical and other industries, and is commonly used as a flavoring agent and valuable spice in food industries. The potential antibiofilm effects of LEO against V. parahaemolyticus were examined in this study. LEO obviously reduced biofilm biomass at subinhibitory concentrations (SICs). It decreased the metabolic activity and viability of biofilm cells. Microscopic images and Raman spectrum indicted that LEO interfered with the structure and biochemical compositions of biofilms. Moreover, it also impaired swimming motility, decreased hydrophobicity, inhibited auto-aggregation and reduced attachment to different food-contact surfaces. RT-qPCR revealed that LEO significantly downregulated transcription levels of biofilm-associated genes of V. parahaemolyticus. These findings demonstrate that LEO could be potentially developed as an antibiofilm strategy to control V. parahaemolyticus biofilms in food industries.

Vibrio splendidus AJ01 Promotes Pathogenicity via L-Glutamic Acid.

Vibrio splendidus is a pathogen that infects a wide range of hosts, especially the sea cucumber species Apostichopus japonicus. Previous studies showed that the level of L-glutamic acid (L-Glu) significantly increased under heat stress, and it was found to be one of the best carbon sources used by V. splendidus AJ01. In this study, the effects of exogenous L-Glu on the coelomocyte viability, tissue status, and individual mortality of sea cucumbers were analyzed. The results showed that 10 mM of L-Glu decreased coelomocyte viability and increased individual mortality, with tissue rupture and pyknosis, while 0.1 mM of L-Glu slightly affected the survival of sea cucumbers without obvious damage at the cellular and tissue levels. Transcriptomic analysis showed that exogenous L-Glu upregulated 343 and downregulated 206 genes. Gene Ontology (GO) analysis showed that differentially expressed genes (DEGs) were mainly enriched in signaling and membrane formation, while a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEGs were significantly enriched in the upregulated endocytosis and downregulated lysosomal pathways. The coelomocyte viability further decreased by 20% in the simultaneous presence of exogenous L-Glu and V. splendidus AJ01 compared with that in the presence of V. splendidus AJ01 infection alone. Consequently, a higher sea cucumber mortality was also observed in the presence of exogenous L-Glu challenged by V. splendidus AJ01. Real-time reverse transcriptase PCR showed that L-Glu specifically upregulated the expression of the fliC gene coding the subunit protein of the flagellar filament, promoting the swimming motility activity of V. splendidus. Our results indicate that L-Glu should be kept in a state of equilibrium, and excess L-Glu at the host-pathogen interface prompts the virulence of V. splendidus via the increase of bacterial motility.

Motility provides specific adhesion patterns and improves Listeria monocytogenes invasion into human HEp-2 cells.

Listeria monocytogenes is motile at 22°C and non-motile at 37°C. In contrast, expression of L. monocytogenes virulence factors is low at 22°C and up-regulated at 37°C. Here, we studied a character of L. monocytogenes near surface swimming (NSS) motility and its effects on adhesion patterns and invasion into epithelial cells. L. monocytogenes and its saprophytic counterpart L. innocua both grown at 22°C showed similar NSS characteristics including individual velocities, trajectory lengths, residence times, and an asymmetric distribution of velocity directions. Similar NSS patterns correlated with similar adhesion patterns. Motile bacteria, including both pathogenic and saprophytic species, showed a preference for adhering to the periphery of epithelial HEp-2 cells. In contrast, non-motile bacteria were evenly distributed across the cell surface, including areas over the nucleus. However, the uneven distribution of motile bacteria did not enhance the invasion into HEp-2 cells unless virulence factor production was up-regulated by the transient shift of the culture to 37°C. Motile L. monocytogenes grown overnight at 22°C and then shifted to 37°C for 2 h expressed invasion factors at the same level and invaded human cells up to five times more efficiently comparatively with non-motile bacteria grown overnight at 37°C. Taken together, obtained results demonstrated that (i) NSS motility and correspondent peripheral location over the cell surface did not depend on L. monocytogenes virulence traits; (ii) motility improved L. monocytogenes invasion into human HEp-2 cells within a few hours after the transition from the ambient temperature to the human body temperature.

A thermo‐resistant and RNase‐sensitive cargo from Giardia duodenalis extracellular vesicles modifies the behaviour of enterobacteria

AbstractExtracellular vesicles (EVs) recently emerged as important players in the pathophysiology of parasitic infections. While the protist parasite Giardia duodenalis can produce EVs, their role in giardiasis remains obscure. Giardia can disrupt gut microbiota biofilms and transform commensal bacteria into invasive pathobionts at sites devoid of colonizing trophozoites via unknown mechanisms. We hypothesized that Giardia EVs could modify gut bacterial behaviour via a novel mode of trans‐kingdom communication. Our findings indicate that Giardia EVs exert bacteriostatic effects on Escherichia coli HB101 and Enterobacter cloacae TW1, increasing their swimming motility. Giardia EVs also decreased the biofilm‐forming ability of E. coli HB101 but not by E. cloacae TW1, supporting the hypothesis that these effects are, at least in part, bacteria‐selective. E. coli HB101 and E. cloacae TW1 exhibited increased adhesion/invasion onto small intestine epithelial cells when exposed to Giardia EVs. EVs labelled with PKH67 revealed colocalization with E. coli HB101 and E. cloacae TW1 bacterial cells. Small RNA sequencing revealed a high abundance of ribosomal RNA (rRNA)‐ and transfer RNA (tRNA)‐derived small RNAs, short‐interfering RNAs (siRNAs) and micro‐RNAs (miRNAs) within Giardia EVs. Proteomic analysis of EVs uncovered the presence of RNA chaperones and heat shock proteins that can facilitate the thermal stability of EVs and its sRNA cargo, as well as protein‐modifying enzymes. In vitro, RNase heat‐treatment assays showed that total RNAs in EVs, but not proteins, are responsible for modulating bacterial swimming motility and biofilm formation. G. duodenalis small RNAs of EVs, but not proteins, were responsible for the increased bacterial adhesion to intestinal epithelial cells induced upon exposure to Giardia EVs. Together, the findings indicate that Giardia EVs contain a heat‐stable, RNase‐sensitive cargo that can trigger the development of pathobiont characteristics in Enterobacteria, depicting a novel trans‐kingdom cross‐talk in the gut.

Inhibitory effect of resveratrol on swimming motility and adhesion ability against Salmonella enterica serovar Typhimurium infection

Salmonella enterica serovar Typhimurium (S. typhimurium) is a common Gram-negative foodborne pathogen that threatens public health and hinders the development of livestock industry. Resveratrol, an important component in grape fruits and seeds, has been shown to possess multiple biological activities, but its potential effects on S. typhimurium-mediated virulence have been rarely reported. In this study, we investigated the effect of resveratrol on S. typhimurium flagella -mediated virulence. The results showed that resveratrol significantly reduced the transcription of flagella genes and swimming motility of S. typhimurium, and also inhibited the transcription of T3SS-related virulence genes with varying degrees inhibiting bacterial growth. Simultaneously, resveratrol significantly reduced the adhesion of S. typhimurium to HeLa cells. Unfortunately, resveratrol does not improve the survival rate of S. typhimurium-infected mice, but it reduces the bacterial load in the liver and spleen of infected mice, and it also has a certain degree of anti-inflammatory activity. In summary, these results indicated that resveratrol has the potential to be developed as an alternative drug or antibacterial agent to prevent Salmonella infection.

Anti-quorum sensing activity of selected cationic amino acids against Chromobacterium violaceum and Pseudomonas aeruginosa

Antibiotic-resistant strains and "superbugs" have emerged as a result of unrestricted use and overuse of antibiotics. N-Acyl homoserine lactones, also known as autoinducers, are unique type of signaling molecule and passively permeate to the cells of Gram-negative bacteria. Accumulation of signaling molecules in the environment regulates the bacterial population. The organisms Pseudomonas aeruginosa and Chromobacterium violaceum are Gram-negative bacteria when exposed to humans, cause fatal infections. The selected cationic amino acids histidine, arginine, and ornithine were assessed for their anti-quorum sensing activity against the P. aeruginosa and C. violaceum. The MIC and sub-MIC concentrations of the selected amino acids have been found by the broth dilution method. Both the cultures have been treated with MIC and sub-MIC levels and the virulence factors like rhamnolipid, protease, elastase, pyoverdine, pyocyanin for P. aeruginosa and violacein, chitinase for C. violaceum is quantified. The quantification of exo-polysaccharides and the biofilm as well as visualization by confocal laser scanning microscope have been done for both organisms. The movement of the cells was assessed by swarming and swimming motility in both organisms. The genes activated for all these virulence factors were quantified by RT-qPCR and expressed in fold change. From the results, it is evident that the histidine inhibited both the organisms at concentrations of MIC and sub-MIC more than the percentage of virulence factors production by arginine and ornithine. The selected cationic amino acids prove to be an effective compound in antimicrobial therapeutics in order to reduce opportunistic bacterial infections.

Antimicrobial and antibiofilm activities of formylchromones against Vibrio parahaemolyticus and Vibrio harveyi.

Gram-negative Vibrio species are major foodborne pathogens often associated with seafood intake that causes gastroenteritis. On food surfaces, biofilm formation by Vibrio species enhances the resistance of bacteria to disinfectants and antimicrobial agents. Hence, an efficient antibacterial and antibiofilm approach is urgently required. This study examined the antibacterial and antivirulence effects of chromones and their 26 derivatives against V. parahaemolyticus and V. harveyi. 6-Bromo-3-formylchromone (6B3FC) and 6-chloro-3-formylchromone (6C3FC) were active antibacterial and antibiofilm compounds. Both 6B3FC and 6C3FC exhibited minimum inhibitory concentrations (MICs) of 20 µg/mL for planktonic cell growth and dose-dependently inhibited biofilm formation. Additionally, they decreased swimming motility, protease activity, fimbrial agglutination, hydrophobicity, and indole production at 20 µg/mL which impaired the growth of the bacteria. Furthermore, the active compounds could completely inhibit the slimy substances and microbial cells on the surface of the squid and shrimp. The most active compound 6B3FC inhibited the gene expression associated in quorum sensing and biofilm formation (luxS, opaR), pathogenicity (tdh), and membrane integrity (vmrA) in V. parahaemolyticus. However, toxicity profiling using seed germination and Caenorhabditis elegans models suggests that 6C3FC may have moderate effect at 50 µg/mL while 6B3FC was toxic to the nematodes 20-100 µg/mL. These findings suggest chromone analogs, particularly two halogenated formylchromones (6B3FC and 6C3FC), were effective antimicrobial and antibiofilm agents against V. parahaemolyticus in the food and pharmaceutical sectors.

Exposure to endocrine disruptors promotes biofilm formation and contributes to increased virulence of Pseudomonas aeruginosa.

Anthropogenic activities contribute to the spread of chemicals considered as endocrine disruptors (ED) in freshwater ecosystems. While several studies have reported interactions of EDs with organisms in those ecosystems, very few have assessed the effect of these compounds on pathogenic bacteria. Here we have evaluated the impact of five EDs found in aquatic resources on the virulence of human pathogen P. aeruginosa. ED concentrations in French aquatic resources of bisphenol A (BPA), dibutyl phthalate (DBP), ethylparaben (EP), methylparaben (MP) and triclosan (TCS) at mean molar concentration were 1.13, 3.58, 0.53, 0.69, and 0.81 nM respectively. No impact on bacterial growth was observed at EDs highest tested concentration. Swimming motility of P. aeruginosa decreased to 28.4% when exposed to EP at 100 μM. Swarming motility increased, with MP at 1 nM, 10 and 100 μM (1.5-fold); conversely, a decrease of 78.5%, with DBP at 100 μM was observed. Furthermore, exposure to 1 nM BPA, DBP and EP increased biofilm formation. P. aeruginosa adhesion to lung cells was two-fold higher upon exposure to 1 nM EP. We demonstrate that ED exposure may simultaneously decrease mobility and increase cell adhesion and biofilm formation, which may promote colonisation and establishment of the pathogen.

Cladosporium sphaerospermum extract inhibits quorum sensing associated virulence factors of Serratia marcescens

Serratia marcescens is now becoming a propensity for its highly antimicrobial-resistant clinical infections. Currently, it provides a novel strategy to prevent and control microbial infection by regulating S. marcescens quorum sensing (QS). Deep-sea-derived fungi are rich in QS bioactive constituents. In this work, the extracts from Cladosporium sphaerospermum SCSGAF0054 showed potent QS-related virulence factors and biofilm-inhibiting activities against S. marcescens NJ01. The swimming motility and multiple virulence factors such as prodigiosin, exopolysaccharide (EPS), lipase, protease and hemolysin were moderately inhibited by the extracts at varied concentrations. The confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) images revealed that C. sphaerospermum extracts moderately arrested biofilm formation and cell viability. Further, real-time quantitative PCR (RT-qPCR) analysis revealed that expressions of genes associated with virulence factors, flhD, fimA, fimC, bsmA, bsmB, pigA, pigC, and shlA, were significantly down-regulated compared with control. In addition, the extracts combined with imipenem inhibited the QS system of S. marcescens NJ01, disrupted its preformed biofilm, released the intra-biofilm bacteria and killed the bacteria gradually. Therefore, the extracts combined with imipenem can partially restore bacterial drug sensitivity. These results suggest that the extracts from SCSGAF0054 effectively interfere with the QS system to treat S. marcescens infection alone or combining with classical antimicrobial drugs.

Flagellar gene fliP contributes to the virulence of Pseudomonas plecoglossicida by regulating its motility

As an opportunistic causative pathogen of “Visceral White Spot Disease” in several kinds of teleost, Pseudomonas plecoglossicida has caused huge economic losses to aquaculture. Flagella is the motility organ of bacteria as well as a vital virulence factor. The protein encoded by the fliP gene is involved in the formation of the flagellar type III secretion export apparatus, which plays a crucial role in flagellar assembly. In this study, the ΔfliP and C-ΔfliP strains of P. plecoglossicida were constructed to explore the biological profiles mediated by the fliP gene and its effects on the virulence of this organism. Compared to the wild type strain (NZBD9) of P. plecoglossicida, the deletion strain ΔfliP exhibited a significant impairment in swimming motility, chemotaxis, adhesion, and biofilm formation abilities. The transcriptomic analysis of the NZBD9, ΔfliP and C-ΔfliP strains revealed that the fliP gene may affect the biological profiles of P. plecoglossicida by down-regulating the expression of flagellar assembly, type 6 secretion system and bacterial chemotaxis related genes. Furthermore, the results of artificial infection showed that the virulence of ΔfliP strain to hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) was attenuated, with a LD50 value of 2.5 × 104 CFU/fish. These findings demonstrate that the fliP gene contributes to the virulence of P. plecoglossicida by regulating its motility.

Biosurfactant derived from probiotic Lactobacillus acidophilus exhibits broad-spectrum antibiofilm activity and inhibits the quorum sensing-regulated virulence.

Antimicrobial resistance by pathogenic bacteria has become a global risk to human health in recent years. The most promising approach to combating antimicrobial resistance is to target virulent traits of bacteria. In the present study, a biosurfactant derived from the probiotic strain Lactobacillus acidophiluswas tested against three Gram-negative bacteria to evaluate its inhibitory potential on their biofilms, and whether it affected the virulence factors controlled by quorum sensing (QS). A reduction in the virulence factors of Chromobacterium violaceum (violacein production), Serratia marcescens (prodigiosin production) and Pseudomonas aeruginosa (pyocyanin, total protease, LasB elastase and LasA protease production) was observed at different sub-MIC concentrations in a dose-dependent manner. Biofilm development was reduced by 65.76%, 70.64% and 58.12% at the highest sub-MIC levels for C. violaceum, P. aeruginosa and S. marcescens, respectively. Biofilm formation on glass surfaces exhibited significant reduction, with less bacterial aggregation and reduced formation of extracellular polymeric materials. Additionally, swimming motility and exopolysaccharides (EPS) production were shown to be reduced in the presence of the L. acidophilus-derived biosurfactant. Furthermore, molecular docking analysis performed on compounds identified through gas chromatography-mass spectrometry (GC-MS) analysis of QS and biofilm proteins yielded further insights into the mechanism underlying the anti-QS activity. Therefore, the present study has clearly demonstrated that a biosurfactant derived from L. acidophilus can significantly inhibit virulence factors of Gram-negative pathogenic bacteria. This could provide an effective method to inhibit the formation of biofilms and QS in Gram-negative bacteria.

The conserved iol gene cluster in Pseudomonas is involved in rhizosphere competence

The Pseudomonas genus has shown great potential as a sustainable solution to support agriculture through its plant-growth-promoting and biocontrol activities. However, their efficacy as bioinoculants is limited by unpredictable colonization in natural conditions. Our study identifies the iol locus, a gene cluster in Pseudomonas involved in inositol catabolism, as a feature enriched among superior root colonizers in natural soil. Further characterization revealed that the iol locus increases competitiveness, potentially caused by an observed induction of swimming motility and the production of fluorescent siderophore in response to inositol, a plant-derived compound. Public data analyses indicate that the iol locus is broadly conserved in the Pseudomonas genus and linked to diverse host-microbe interactions. Together, our findings suggest the iol locus as a potential target for developing more effective bioinoculants for sustainable agriculture.

Ginger-Enriched Honey Attenuates Antibiotic Resistant Pseudomonas aeruginosa Quorum Sensing Virulence Factors and Biofilm Formation.

Quorum sensing (QS) in Pseudomonas aeruginosa plays an essential role in virulence factors, biofilm formation as well as antibiotic resistance. Approaches that target virulence factors are known to be more sustainable than antibiotics in weakening the infectivity of bacteria. Although honey has been shown to exert antipseudomonal activities, the enhancement of such activity in ginger-enriched honey is still unknown. The main objective of this study was to determine the impacts of honey and ginger-enriched honey on the QS virulence factors and biofilm formation of antibiotic resistant P. aeruginosa clinical isolates. Outcomes showed honey and/or ginger-enriched honey significantly reduced the protease activity, pyocyanin production and exotoxin A concentration of the isolates. The swarming and swimming motility together with biofilm formation in all clinical isolates were also significantly inhibited by both honey samples. Notable morphological alteration of bacterial cells was also observed using scanning electron microscopy. A principal component analysis (PCA) managed to distinguish the untreated group and treatment groups into two distinct clusters, although honey and ginger-enriched honey groups were not well differentiated. This study revealed the effectiveness of honey including ginger-enriched honey to attenuate QS virulence factors and biofilm formation of P. aeruginosa.