ToxR Is Required for Biofilm Formation and Motility of Vibrio Parahaemolyticus

Vibrio parahaemolyticus, the leading cause of seafood-borne gastroenteritis, has the ability to form biofilms on biotic and abiotic surfaces. Biofilm formation is a complicated process involving many specific structures and regulatory processes. The most significant of the structures and processes include polar and lateral flagella, mannose-sensitive hemagglutinin typeⅣpili, chitin-regulated pili,capsular polysaccharide (CPS), exopolysaccharide

Anti-quorum sensing and anti-biofilm formation activities of plant extracts from South Korea

Objective: To investigate anti-quorum sensing(anti-QS) and anti-biofilm formation(antiBF) activities of the ethanol extracts of 388 plants. Methods: The anti-QS activity of the plant extracts was evaluated by disc-diffusion assays using the bio-reporter strain, Chromobacterium violaceum CV017. Pseudomonas aeruginosa PAO1, Yersinia enterocolitica ATCC 9610, and Agrobacterium tumefaciens C58, which possess QS systems, were used to evaluate the antiBF activity of the plant extracts. Results: Among 388 plant extracts, the Cornus controversa(C. controversa) and Cynanchum wilfordii extracts exhibited the strongest anti-QS activity. The C. controversa extract exhibited anti-BF activity against Pseudomonas aeruginosa, Yersinia enterocolitica and Agrobacterium tumefaciens, whereas the Cynanchum wilfordii extract exhibited no anti-BF activity against Pseudomonas aeruginosa. In addition, the C. controversa extract suppressed soft rot of cabbage. Conclusions: The C. controversa extract inhibits bacterial QS and BF, and is capable of controlling soft rot. Therefore, this extract has potential for the prevention and treatment of bacterial infections and for the development of alternatives to antibiotics.

Identification of the nitrogen-fixing Pseudomonas stutzeri major flagellar gene regulator FleQ and its role in biofilm formation and root colonization

Flagellar biosynthesis and motility are subject to a four-tiered transcriptional regulatory circuit in Pseudomonas,and the master regulator FleQ appears to be the highest-level regulator in this hierarchical regulatory cascade.Pseudomonas stutzeri A1501 is motile by a polar flagellum;however,the motility and regulatory mechanisms involved in this process are unknown.Here,we searched the A1501 genome for flagella and motility genes and found that approximately 50 genes,which were distributed in three non-contiguous chromosomal regions,contribute to the formation,regulation and function of the flagella.The non-polar mutation of fleQ impaired flagellar biosynthesis,motility and root colonization but enhanced biofilm formation.FleQ positively regulates the expression of flagellar class Ⅱ-Ⅳ genes,suggesting a regulatory cascade that is coordinated similar to that of the well-known P.aeruginosa.Based on our results,we propose that flagellar genes in P.stutzeri A1501 are regulated in a cascade regulated by FleQ and that flagellum-driven motility properties may be necessary for competitive rhizosphere colonization.

In vitro biofilm formation in ESBL producing Escherichia coli isolates from cage birds

Objective:To determine biofilm and hydrophobicity formation ratios in extended spectrum beta lactamases(ESBL) synthesizing Escherichia coli isolates which were isolated from feces samples of 150 cage bird species randomly taken from pet shops in Hatay province,Turkey.Methods:In vitro biofilm production of 4 ESBL positive isolates were performed by Congo Red Agar(CRA),Standard Tube(ST) and Microtitre Plate(MP) methods while their hydrophobicity were examined by bacterial adhesion to hydrocarbon(BATH) test.Results:In the examined isolates,while biofilm production was found to be negative by CRA method,highest biofilm producing strain,among 4 bacteria was determined to be A42 by ST and MP methods.The Scanning Electron Microscopy(SEM) also displayed these confirmed findings.The hydrophobicity values of strains were determined to be between 22.45%and 26.42%.Conclusions:As a result,biofilm formation in cage bird feces originated ESBL positive Escherichia coli isolates was performed for the first time in Turkey.In order to present the relation between pathogenicity and biofilm production in animal originated ESBL positive isolates,further studies are required.

Study of Biofilm Formation and Antibiotic Resistance Pattern of Bacteria Isolated from Diabetic Foot Ulcers in Hôpital de Référence Saint Joseph, Kinshasa, Democratic Republic of Congo

Foot infections resulting from biofilm producers and multi-drug resistant organisms is one of the most important complications of diabetes mellitus, as it can impede the wound healing process. This study was carried out in order to determine the antibiotic resistance pattern and the biofilm production in diabetic foot ulcers isolates. Clinical samples were collected from patients suffering from diabetic foot ulcers by using sterile swabs. Antibiotic susceptibility test was done using disk diffusion method on Mueller Hinton Agar. Biofilm formation was assessed by Crystal Violet Staining Method. Staphylococcus aureus isolates were resistant to ofloxacin (83.3%), ciprofloxacin (75.0%), trimethoprim-sulphamethoxazole (75.0%), and gentamicin (58.8%) but very sensitive to oxacillin (100.0%) and vancomycin (91.7%). Pseudomonas aeruginosa isolates showed resistance to the commonly used antibiotics such as ofloxacin, cefotaxime, ampicillin (81.8%), ceftazidime and imipenem (72.7%). The majority of bacteria studied were biofilm producers. This study showed that bacteria isolated from diabetic foot ulcers were biofilm producers and presented resistance to commonly used antibiotics. Knowledge on antibiotic sensitivity pattern and biofilm phenotype of the isolates will be helpful in determining the drugs for the treatment of diabetic ulcers.

Effects of quorum sensing autoinducer degradation gene on virulence and biofilm formation of Pseudomonas aeruginosa

The aiiA gene from Bacillus thuringiensis was cloned into the Pseudomonas/E. coli shuttle vector and transformed into Pseudomonas aeruginosa strain PAO1. Western blotting showed that the AiiA protein was expressed in PAO1. After induction by IPTG for 6 h and 18 h, expression of the aiiA gene in PAO1 completely degraded the quorum sensing autoinducers N-acylhomoserine lactones (AHLs): N-oxododecanoyl-L-homoserine lactone (OdDHL) and N-butyryl-L-homoserine lactone (BHL). The re- duced amount of AHLs in PAO1 was also correlated with decreased expression and production of several virulence factors such as elastase and pyocyanin. AiiA expression also influenced bacterial swarming motility. Most importantly, our studies indicated that aiiA played significant roles in P. aeruginosa biofilm formation and dispersion, as observed by the differences of the biofilm formation on liquid and solid surfaces, and biofilm structures under a scanning electron microscope.

Bioactive Titanium Surfaces with the Effect of Inhibiting Biofilm Formation

Titanium metals and its alloy have been widely used in hard tissue repairing fields due to their good biocampatibility and mechanical properties. However, bioinert response and biomaterial associated infections are the main problems for their clinical application. In this study, we chose titanium plates treated with anodic oxidation （AO-Ti）, alkali-heat （AH-Ti） and acid-alkali （AA-Ti） methods, which have been proved to be bioactive in vivo, to culture with Staphylococcus aureus and Escherichia coli to investigate the interaction between bioactive titanium surfaces and biofilm. We used X-ray diffraction （XRD）, Scanning Electron Microscope （SEM）, roughness measurement to study the physical-chemical properties of the as-received bioactive titanium surfaces, and Confocal Laser Scanning Microscope （CLSM） was employed to study the properties of biofilm formed on the biomaterial surfaces. The results indicate that the titanium surface subjected to anodic oxidation treatment is unfavorable for the formation ofbiofilm in vitro because the titania （TiO2） coating formed by anodizing has superior antimicrobial property than the other surfaces. Therefore, anodic oxidation surface modification is effective to endow titanium surface with bioactivity and antimicrobial property, which has the potential to improve the successful rate of the clinical application of titanium implants.

Characterization of a novel regulatory pathway for mannitol metabolism and its coordination with biofilm formation in Mycobacterium smegmatis

Biofilm formation has been implicated to be tightly regulated in bacteria. Mycobacterial species possess a unique cell-wall structure; however, the underlying regulation mechanism for their biofilm formation remains largely unclear. In this study, we characterized a hypothetical mannitol metabolism and transportation gene cluster（Ms5571-Ms5576）, designated as mmt operon, whose expression significantly contributes to the biofilm formation in Mycobacterium smegmatis. We showed that in the operon the Ms5575 gene encodes a GntR-like transcriptional repressor and the Ms5576 gene encodes a mannitol2-dehydrogenase which can produce D-mannitol from D-mannose. Strikingly, the D-mannitol molecule can derepress the negative regulation of Ms5575 on the mmt operon to stimulate the operon＇s expression. Consistently, addition of D-mannitol into the medium can obviously induce mycobacterial biofilm formation. Furthermore, we found that Ms0179 positively regulates the mmt operon through its downstream regulator Ms0180. Ms0180 directly binds the mmt operon to positively regulate its expression. Both Ms0179 and Ms0180 significantly affect the mycobacterial biofilm formation. Taken together, we explored a regulatory pathway for the mannitol metabolism and its coordination with the biofilm formation in M. smegmatis. This finding provides novel insights into the unique mechanism of biofilm formation regulation in mycobacteria.

Mechanistic insight into the biofilm formation and process performance of a passive aeration ditch(PAD)for decentralized wastewater treatment

Decentralized wastewater containing elevated salinity is an emerging threat to the local environment and sanitation in remote coastal communities.Regarding the cost and treatment efficiencies,we propose a passive aeration ditch(PAD)using non-woven polyester fabric as a feasible bubbleless aerator and biofilm carrier for wastewater treatment.Consideration has been first given to PAD’s efficacy in treating saline decentralized wastewater,and then to the impact of chemical oxygen demand-to-nitrogen(C/N)ratio and salinity on biofilm formation.A multispecies model incorporating the salinity effect has been developed to depict the system performance and predict the microbial community.Results showed that the PAD system had great capacity for pollutants removal.The biofilm thickness increased at a higher C/N ratio because of the boost of aerobic heterotrophs and denitrifying bacteria,which consequently improved the COD and total nitrogen removal.However,this led to the deterioration of ammonia removal.Moreover,while a higher salinity benefited the biofilm growth,the contaminant removal efficiencies decreased because the salinity inhibited the activity of aerobic heterotrophs and reduced the abundance of nitrifying bacteria inside the biofilm.Based on the model simulation,feed water with salinity below 2%and C/N ratio in a range of 1 to 3 forms a biofilm that can reach relatively high organic matter and ammonia removal.These findings not only show the feasibility of PAD in treatment of saline decentralized wastewater,but also offer a systematic strategy to predict and optimize the process performance.

NEW STRATEGIES FOR THE CONTROL OF BACTERIAL INFECTIONS: MODULATORS OF QUORUM SENSING AND BIOFILM FORMATION FROM MALAGASY DALBERGIA SPECIES

Considering the WHO warning about the emergence of a’post-antibiotic’era during the 21st century in which common infections and minor injuries will have a dramatic impact on human death toll,search for new potential antibacterial drug targets became a necessary need.Targets that are extensively explored concern the modulation

Cell Surface Determinants Important for Biofilm-Based Solid Substrate Degradation

The study links targeted cell surface characterization to the quantified capacity of cellulose degrading Pseudomonas fluorescens cells to colonize a (similarly characterized) cellulosic carrier. The experiments were conducted to clarify the effect of cultivation conditions on the achieved state of this carrier colonization. The suggested approach seems to be sufficient to verify the right choice of cultivation medium as a major factor determining the binding complementarity between microbial cells and solid cellulose.

Investigation of Pb(Ⅱ)bioremediation potential of algae and cyanobacteria strains isolated from polluted water

Algae and cyanobacteria are known to be able to remove a variety of pollutants from water,including toxic metal ions.In this study,algal and cyanobacterial species growing in two polluted water bodies were identified,and the Pb(Ⅱ)removal ability of these isolated species was investigated.Based on microscopic observations,27 species were identified,and nine species were isolated as pure cultures.Pb(Ⅱ)bioremediation of five selected species(Anacystis sp.,Chlorella sp.1,Monoraphidium sp.,Phormidium sp.,and Uronema sp.)was studied in detail.The mean Pb(Ⅱ)removal abilities varied among these five species:Phormidium sp.>Monoraphidium sp.>Uronema sp.>Chlorella sp.1>Anacystis sp.The Pb(Ⅱ)tolerance of each species was determined based on the live cell percentage and biofilm formation capacity.Within a period of 3 d,Phormidium sp.,Monoraphidium sp.,and Uronema sp.showed nearly 90%of survival,and all five species demonstrated biofilm formation capacities exceeding 50%.Furthermore,the Pb(Ⅱ)removal ability of the five species exhibited a strong positive correlation with the live cell percentage and showed a strong negative correlation with the biofilm formation capacity.In conclusion,Phormidium sp.,Monoraphidium sp.,and Uronema sp.exhibited high tolerances towards Pb(Ⅱ)and presented high removal abilities.Thus,these species can be identified as potential sorbents for development of suitable adsorption systems to remove Pb(Ⅱ)from aqueous solutions.

Effects of exogenous acylated homoserine lactones on biofilms in biofilters for gaseous toluene treatment

Biofilters are typical biofilm reactors, and they usually have poor biofilm formation resulting in limited reactor performance. Exogenous acylated homoserine lactones (AHLs) can enhance biofilm formation in many bioreactors based on quorum sensing regulation. However, their effect on biofilm in biofilters utilized for volatile organic compound (VOC) removal is unknown and needs to be investigated. In this study, the effects of the exogenous AHLs on biofilters for gaseous toluene removal were investigated. Analysis of biofilms in biofilters showed that the addition of exogenous AHLs considerably enhanced biofilm growth;the average biofilm concentration increased by 18%. Furthermore, the average biofilm coverage proportions in biofilters with and without exogenous AHLs were 17 % and 13 %, respectively, demonstrating the positive effect of exogenous AHLs on biofilm coverage. In particular, exogenous AHLs promoted the production of extracellular polymeric substances and the microbial adhesive strength of the biofilm. In addition, the exogenous AHLs showed no significant effect on the gaseous toluene removal efficiency of the biofilter. These results show that exogenous AHLs can enhance biofilm formation and can guide the application of exogenous AHLs in VOC biofilters.

Influence and mechanism of N-(3-oxooxtanoyl)-L-homoserine lactone(C_8-oxo-HSL) on biofilm behaviors at early stage

N-acyl-homoserines quenching, enzymatic quenching of bacterial quorum sensing, has recently applied to mitigate biofilm in membrane bioreactor. However, the effect of AHLs on the behavior of biofilm formation is still sparse. In this study, Pseudomonas aeruginosa biofilm was formed on ultra-filtration membrane under a series of N-（3-0xooxtanoyl）-L-homoserine lactone （C8-0xo-HSL） concentrations. Diffusing Cs-oxo-HSL increased the growth rate of cells on biofilm where the concentration of Cs-oxo-HSL was over 10-7 g/L. The C8-0xo-HSL gradient had no observable influence on cell density and extracellular polymeric substances of biofilm with over 10-7 g/L Cs-oxo-HSL. Surprisingly, 10-11-10-8 g/L of Cs-Oxo-HSL had no effect on cell growth in liquid culture. The cell analysis demonstrated that the quorum sensing system might enhance the growth of neighboring cells in contact with surfaces into biofilm and may influence the structure and organization of biofilm.

Biofilm-overproducing Bacillus subtilis B12ΔYwcc decreases Cd uptake in Chinese cabbage through increasing Cd-immobilizing related gene abundance and root surface colonization

Biofilm-producing bacteria can decrease Cd uptake in vegetables, but mechanisms underlying this effect are poorly characterized. In this study, two mutant strains B12ΔYwcc and B12ΔSlr R were constructed from a biofilm-producing Bacillus subtilis strain B12. Then, the impacts of strain B12 and its high biofilm-producing mutant strain B12ΔYwcc and low biofilmproducing mutant strain B12ΔSlr R on Cd availability and uptake in Chinese cabbage and the related mechanisms were investigated in the Cd-polluted soil. Strain B12 and its mutants B12ΔYwcc and B12ΔSlr R increased the dry biomasses of edible tissues by 54%–130% compared with the controls. Strain B12 and its mutant B12ΔYwcc reduced the soil available Cd content by 36%–50% and root and edible tissue Cd contents by 23%–50% compared with the controls. Furthermore, the mutant strain B12ΔYwcc reduced the edible tissue Cd content by40% and increased the polysaccharide content by 23%, invertase activity by 139%, and gene copies of the cum A by 4.5-fold, eps A by 7.1-fold, and cad A by 4.3-fold, which were involved in Cd adsorption in the rhizosphere soils, respectively, compared with strain B12. The polysaccharide content and cum A, eps A, and cad A gene copy numbers showed significantly reverse correlations with the available Cd content. Notably, the mutant strain B12ΔYwcc showed better ability to colonize the vegetable root surface than strain B12. These findings demonstrated that the biofilm-overproducing mutant strain B12ΔYwcc increased the polysaccharide production and Cd-immobilizing related cum A, eps A, and cad A gene copies, resulting in lower Cd availability and accumulation in Chinese cabbage in the Cd-polluted soil.

Quorum sensing regulation methods and their effects on biofilm in biological waste treatment systems: A review

Quorum sensing (QS) plays an important role in microbial aggregation control. Recently, the optimization of biological waste treatment systems by QS regulation gained an increasing attention. The effects of QS regulation on treatment performances and biofilm were frequently investigated. To understand the state of art of QS regulation, this review summarizes the methods of QS enhancement and QS inhibition in biological waste treatment systems. Typical QS enhancement methods include adding exogenous QS molecules, adding QS accelerants and cultivating QS bacteria, while typical QS inhibition methods include additions of quorum quenching (QQ) bacteria, QS-degrading enzymes, QS-degrading oxidants, and QS inhibitors. The specific improvements after applying these QS regulation methods in different treatment systems are concluded. In addition, the effects of QS regulation methods on biofilm in biological waste treatment systems are reviewed in terms of biofilm formation, extracellular polymeric substances production, microbial viability, and microbial community. In the end, the knowledge gaps in current researches are analyzed, and the requirements for future study are suggested.

Accelerated neutral atom beam(ANAB)and gas clustered ion beam(GCIB)treatment of implantable device polymers leads to decreased bacterial attachment in vitro and decreased inflammation in vivo

Infections at the placement site of biomaterial-based devices and subsequent scar formation results in morbidity,which may require revision surgery.Biomaterials intended for permanent implantation in the body need to be biologically inert to avoid excessive foreign body response and to reduce bacterial attachment.In this study,we show that polymeric materials commonly used in medical devices,including polyetheretherketone(PEEK)and polypropylene,treated by gas cluster ion beam(GCIB)or by accelerated neutral atom beam(ANAB)result in a nanoscale-modified surface topography that changes the ability of extracellular proteins to bind.This leads to decreased bacterial attachment and an attenuated inflammatory response using both in vitro and in vivo assays.Differential adsorption of extracellular proteins to the polymeric surface improved the competitive attachment of osteoblasts over bacteria,without resorting to growth factor of antibiotic use.

Clinical and microbiological characterization of Clostridium difficile infection in a tertiary care hospital in Shanghai, China

Background Over the last decade,Clostridium difficile infection （CDI） has emerged as a significant nosocomial infection,yet little has been reported from China.This study aimed to characterize the clinical and microbiological features of CDI from a hospital in Shanghai.Methods Patients with CDI seen between December 2010 and March 2013 were included in this study,of which clinical data were retrospectively collected.The microbiological features of corresponding isolates were analyzed including genotype by multi-locus sequence typing （MLST）,antimicrobial susceptibility,toxin production,sporulation capacity,biofilm formation,and motility.Results Ninety-four cases of CDI were included during this study period,12 of whom were severe cases.By reviewing the clinical data,all patients were treated empirically with proton pump inhibitor or antibiotics or both,and they were distributed widely across various wards,most frequently to the digestive ward （28/94,29.79％）.Comparing the severe with mild cases,no significant differences were found in the basic epidemiological data or the microbiological features.Among the 94 isolates,31 were toxin A-negative toxin B-positive all genotyped as ST37.They generated fewer toxins and spores,as well as similar amounts of biofilm and motility percentages,but exhibited highest drug resistance to cephalosporins,quinolones,macrolide-lincosamide and streptogramin （MLSB）,and tetracycline.Conclusions No specific clinical genotype or microbiological features were found in severe cases; antimicrobial resistance could be the primary reason for epidemic strains leading to the dissemination and persistence of CDI.

A new iron(Ⅲ) chelator of coprogen-type siderophore from the deep-sea-derived fungus Mycosphaerella sp. SCSIO z059

Mycosphazine A(1), a new iron(Ⅲ) chelator of coprogen-type siderophore, and mycosphamide A(2), a new cyclic amide benzoate, together with six known aryl amides(3-8), were isolated from the fermentation broth of the deep-sea-derived fungus Mycosphaerella sp. SCSIO z059. Alkaline hydrolysis of 1 afforded a new epimer of dimerum acid, mycosphazine B(1 a), and a new bi-fusarinine-type siderophore, mycosphazine C(1 b). The planar structures of the new compounds were elucidated by extensive spectroscopic data analysis. The absolute configurations of amino acid residues in 1 a and 1 b were determined by acid hydrolysis. And the absolute configuration of 2 was established by quantum chemical calculations of the electronic circular dichroism(ECD) spectra. Compound 1 is the first siderophore-Fe(Ⅲ) chelator incorporating both L-ornithine and D-ornithine unites. Compounds 3-8 were reported as natural products for the first time, and the 1 H and 13 C NMR data of 6 and 8 were assigned for the first time. Compounds 1 and 1 a could greatly promote the biofilm formation of bacterium Bacillus amyloliquefaciens with the rate of about 249% and 524% at concentration of 100 μg·mL-1, respectively.

Characterization of the archaeal community fouling a membrane bioreactor

Biofilm formation, one of the primary causes of biofouling, results in reduced membrane flux or increased transmembrane pressure and thus represents a major impediment to the wider implementation of membrane bioreactor （MBR） technologies for water purification. Most studies have focused on the role of bacteria in membrane fouling as they are the most dominant and best studied organisms present in the MBR. In contrast, there is limited information on the role of the archaeal community in biofilm formation in MBRs. This study investigated the composition of the archaeal community during the process of biofouling in an MBR. The archaeal community was observed to have lower richness and diversity in the biofilm than the sludge during the establishment ofbiofilms at low transmembrane pressure （TMP）. Clustering of the communities based on the Bray-Curtis similarity matrix indicated that a subset of the sludge archaeal community formed the initial biofilms. The archaeal community in the biofilm was mainly composed of Thermoprotei, Thermoplasmata, Thermococci, Methanopyri, Methanomicrobia and Halobacteria. Among them, the Thermoprotei and Thermoplasmata were present at higher relative proportions in the biofilms than they were in the sludge. Additionally, the Thermoprotei, Thermoplasmata and Thermococci were the dominant organisms detected in the initial biofilms at low TMP, while as the TMP increased, the Methanopyri, Methanomicrobia, Aciduliprofundum and Halobacteria were present at higher abundances in the biofilms at high TMP.