Investigating the effect of patulin,penicillic acid and EDTA on biofilm formation of isolates from dental unit water lines

This study investigated the effect of patulin and penicillic acid, two known quorum-sensing inhibitors, and the common biocide ethylenediaminetetraacetic acid (EDTA) on the biofilm formation and auto-inducer (AI)-2 production of three isolates from dental unit water lines, Klebsiella sp., Bacillus subtilis and Bacillus cereus. Penicillic acid on its own had no effect on the biofilm formation of all isolates, whereas in combination with EDTA, it enhanced biofilm formation significantly in Klebsiella sp. and B. cereus. EDTA at concentrations greater than 10 μM promoted biofilm formation in B. cereus and B. subtilis. Patulin was found to promote biofilm formation in B. cereus up to 25 μM. A significant increase in biofilm formation was observed in B. cereus and B. subtilis at concentrations greater than 10 μM of patulin when combined with EDTA. The Vibrio harveyi BB170 AI-2 bioassay showed a positive response for Klebsiella sp. AI-2 production with a maximum fold induction at the late exponential growth phase. Addition of glucose prolonged the AI-2 production phase considerably. No significant effect of patulin, penicillic acid alone as well as in combination with EDTA was observed on AI-2 production by Klebsiella sp. The findings have important implications for the design of biofilm prevention and eradication strategies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Changes in physico-chemical characteristics and viable bacterial communities during fermentation of alfalfa silages inoculated with Lactobacillus plantarum

Sulfate-reducing bacteria (SRB) are culprits for microbiologically influenced corrosion, and biofilms are believed to play essential roles in the corrosion induced by SRB. However, little is known about the regulation of SRB biofilms. Quorum sensing signal molecules acyl-homoserine lactones (AHLs) and autoinducer-2 (AI-2) regulate biofilm formation of many bacteria. In this study, the production of AHLs and AI-2 by one SRB strain, Desulfovibrio sp. Huiquan2017, was detected, and the effect of exogenous AI-2 on bacterial biofilm formation was discussed. It was found that the cell-free supernatants of Desulfovibrio sp. Huiquan2017 induced luminescence in a ?luxS mutant strain Vibrio harveyi BB170, indicating the production of functional AI-2 by the bacterium. In the presence of exogenous AI-2, the growth of Desulfovibrio sp. Huiquan2017 and early biofilm formation were not affected, but the later stage of biofilm development was inhibited significantly. The biofilms became looser, smaller, and thinner, and contained less bacteria and extracellular polymeric substances (EPS). The inhibition effect of AI-2 on the biofilm development of Desulfovibrio sp. Huiquan2017 was mainly achieved through reducing the amount of EPS in biofilms. These findings shed light on the biofilm regulation of SRB.

     

Tetracycline and Chloramphenicol Efficiency Against Selected Biofilm Forming Bacteria

Despite the constantly increasing need for new antimicrobial agents, antibiotic drug discovery and development seem to have greatly decelerated in recent years. Presented with the significant problem of advancing antimicrobial resistance, the global scientific community has attempted to find alternative solutions; one of the most promising ones is the evaluation and use of old antibiotic compounds. A number of old antibiotic compounds, such as aminoglycosides, chloramphenicol, and tetracycline, are re-emerging as valuable alternatives for the treatment of difficult-to-treat infections. This study examined the in vitro potency for biofilm formation of five isolates (Klebsiella sp., Pseudomonas aeruginosa, Achromobacter sp., Klebsiella pneumoniae, and Bacillus pumilis) and the effects of antibiotics on these biofilms. Furthermore the quantitative analysis of planktonic, loosely attached cells, and their susceptibility to antibiotics was also determined. Twitching motility was observed to determine any effect in the biofilm forming capability of the isolates. All the isolates tested were efficient biofilm-forming strains in the polypropylene and borosilicate test tubes. Standard bacterial enumeration technique and CV staining produced equivalent results both in biofilm and planktonic assays. The biofilm formation of all the strains was affected in the presence of tetracycline or chloramphenicol. Highly significant decrease (P < 0.01) in biofilm formation was observed by treatment with chloramphenicol compared to tetracycline. In addition, the two antibiotics also affected adversely the planktonic and loosely attached cells of all isolates. Thus, testing the effects of older antibiotics on biofilms may supply useful information in addition to standard in vitro testing, particularly in diseases where biofilm formation is involved in the pathogenesis.     

Pseudomonas aeruginosa and Achromobacter sp. Clonal Selection Leads to Successive Waves of Contamination of Water in Dental Care Units

Dental care unit waterlines (DCUWs) consist of complex networks of thin tubes that facilitate the formation of microbial biofilms. Due to the predilection toward a wet environment, strong adhesion, biofilm formation, and resistance to biocides, Pseudomonas aeruginosa, a major human opportunistic pathogen, is adapted to DCUW colonization. Other nonfermentative Gram-negative bacilli, such as members of the genus Achromobacter, are emerging pathogens found in water networks. We reported the 6.5-year dynamics of bacterial contamination of waterlines in a dental health care center with 61 dental care units (DCUs) connected to the same water supply system. The conditions allowed the selection and the emergence of clones of Achromobacter sp. and P. aeruginosa characterized by multilocus sequence typing, multiplex repetitive elements-based PCR, and restriction fragment length polymorphism in pulsed-field gel electrophoresis, biofilm formation, and antimicrobial susceptibility. One clone of P. aeruginosa and 2 clones of Achromobacter sp. colonized successively all of the DCUWs: the last colonization by P. aeruginosa ST309 led to the closing of the dental care center. Successive dominance of species and clones was linked to biocide treatments. Achromobacter strains were weak biofilm producers compared to P. aeruginosa ST309, but the coculture of P. aeruginosa and Achromobacter enhanced P. aeruginosa ST309 biofilm formation. Intraclonal genomic microevolution was observed in the isolates of P. aeruginosa ST309 collected chronologically and in Achromobacter sp. clone A. The contamination control was achieved by a complete reorganization of the dental health care center by removing the connecting tubes between DCUs.     

Differential Gene Expression for Investigation of Escherichia coli Biofilm Inhibition by Plant Extract Ursolic Acid

After 13,000 samples of compounds purified from plants were screened, a new biofilm inhibitor, ursolic acid, has been discovered and identified. Using both 96-well microtiter plates and a continuous flow chamber with COMSTAT analysis, 10 μg of ursolic acid/ml inhibited Escherichia coli biofilm formation 6- to 20-fold when added upon inoculation and when added to a 24-h biofilm; however, ursolic acid was not toxic to E. coli, Pseudomonas aeruginosa, Vibrio harveyi, and hepatocytes. Similarly, 10 μg of ursolic acid/ml inhibited biofilm formation by >87% for P. aeruginosa in both complex and minimal medium and by 57% for V. harveyi in minimal medium. To investigate the mechanism of this nontoxic inhibition on a global genetic basis, DNA microarrays were used to study the gene expression profiles of E. coli K-12 grown with or without ursolic acid. Ursolic acid at 10 and 30 μg/ml induced significantly (P < 0.05) 32 and 61 genes, respectively, and 19 genes were consistently induced. The consistently induced genes have functions for chemotaxis and mobility (cheA, tap, tar, and motAB), heat shock response (hslSTV and mopAB), and unknown functions (such as b1566 and yrfHI). There were 31 and 17 genes repressed by 10 and 30 μg of ursolic acid/ml, respectively, and 12 genes were consistently repressed that have functions in cysteine synthesis (cysK) and sulfur metabolism (cysD), as well as unknown functions (such as hdeAB and yhaDFG). Ursolic acid inhibited biofilms without interfering with quorum sensing, as shown with the V. harveyi AI-1 and AI-2 reporter systems. As predicted by the differential gene expression, deleting motAB counteracts ursolic acid inhibition (the paralyzed cells no longer become too motile). Based on the differential gene expression, it was also discovered that sulfur metabolism (through cysB) affects biofilm formation (in the absence of ursolic acid).     

A Mutation in the luxS Gene Influences Listeria monocytogenes Biofilm Formation

Using a Vibrio harveyi reporter strain, we demonstrated that Listeria monocytogenes secretes a functional autoinducer 2 (AI-2)-like signal. A luxS-deficient mutant produced a denser biofilm and attached to a glass surface 19-fold better than the parent strain. Exogenous AI-2 failed to restore the wild-type phenotype to the mutant. It seems that an intact luxS gene is associated with repression of components required for attachment and biofilm formation.     

AI-2通过甲基化趋化受体McpU调控恶臭假单胞菌KT2440的趋化运动及生物膜形成

[背景]广泛存在于革兰氏阴性菌和革兰氏阳性菌中的自诱导物autoinducer-2 (AI-2)能够介导细菌种内和种间通讯,并调节细菌的多种生理过程.然而恶臭假单胞菌KT2440能否感知AI-2信号还未见报道.[目的]挖掘介导恶臭假单胞菌KT2440对AI-2趋化反应的趋化受体,检测AI-2信号通过趋化受体对恶臭假单胞...     

Cinnamaldehyde and cinnamaldehyde derivatives reduce virulence in <Emphasis Type="Italic">Vibrio</Emphasis> spp. by decreasing the DNA-binding activity of the quorum sensing response regulator LuxR

Background  

To date, only few compounds targeting the AI-2 based quorum sensing (QS) system are known. In the present study, we screened cinnamaldehyde and substituted cinnamaldehydes for their ability to interfere with AI-2 based QS. The mechanism of QS inhibition was elucidated by measuring the effect on bioluminescence in several Vibrio harveyi mutants. We also studied in vitro the ability of these compounds to interfere with biofilm formation, stress response and virulence of Vibrio spp. The compounds were also evaluated in an in vivo assay measuring the reduction of Vibrio harveyi virulence towards Artemia shrimp.     

D-Galactose as an autoinducer 2 inhibitor to control the biofilm formation of periodontopathogens

Autoinducer 2 (AI-2) is a quorum sensing molecule to which bacteria respond to regulate various phenotypes, including virulence and biofilm formation. AI-2 plays an important role in the formation of a subgingival biofilm composed mostly of Gram-negative anaerobes, by which periodontitis is initiated. The aim of this study was to evaluate D-galactose as an inhibitor of AI-2 activity and thus of the biofilm formation of periodontopathogens. In a search for an AI-2 receptor of Fusobacterium nucleatum, D-galactose binding protein (Gbp, Gene ID FN1165) showed high sequence similarity with the ribose binding protein (RbsB), a known AI-2 receptor of Aggregatibacter actinomycetemcomitans. D-Galactose was evaluated for its inhibitory effect on the AI-2 activity of Vibrio harveyi BB152 and F. nucleatum, the major coaggregation bridge organism, which connects early colonizing commensals and late pathogenic colonizers in dental biofilms. The inhibitory effect of D-galactose on the biofilm formation of periodontopathogens was assessed by crystal violet staining and confocal laser scanning microscopy in the absence or presence of AI-2 and secreted molecules of F. nucleatum. D-Galactose significantly inhibited the AI-2 activity of V. harveyi and F. nucleatum. In addition, D-galactose markedly inhibited the biofilm formation of F. nucleatum, Porphyromonas gingivalis, and Tannerella forsythia induced by the AI-2 of F. nucleatum without affecting bacterial growth. Our results demonstrate that the Gbp may function as an AI-2 receptor and that galactose may be used for prevention of the biofilm formation of periodontopathogens by targeting AI-2 activity.     

Effect of Biocides on Biofilm Bacteria from Dental Unit Water Lines

Microbial biofilm formation in dental unit water lines (DUWL) is a phenomenon that has been recognized for nearly four decades. Water delivered by DUWL can harbor high numbers of bacteria, including opportunistic pathogens. Biofilms on tubing within DUWL may serve as a reservoir for these microorganisms and should therefore be controlled. In this study, the effects of eight biocides were monitored on DUWL biofilms individually and in combination by epifluorescence microscopy and total viable counts (TVC). The effects of sodium dodecyl sulphate (SDS), hydrogen peroxide (H₂O₂), sodium hypochlorite (NaOCl), phenol (Phe), Tween 20 (Tw 20), ethylenediaminetetraacetic acid (EDTA), chlorohexidine gluconate (CHX), and povidine iodine (PI) were tested on DUWL biofilms alone and in combination. PI was found to have negligible effects on biofilm removal either applied alone or in combined form with CHX. Applying all biocides simultaneously did not completely eliminate viable bacteria nor did they remove biofilm. Overall, when combined, the biocides performed better than singly applied products. The most effective biocides were NaOCl and Phe (both alone and in combination).     

Quorum Sensing Circuit and Reactive Oxygen Species Resistance in Deinococcus sp.

Genus Deinococcus is characterized by an increased resistance toward reactive oxygen species (ROS). The chromosome of five strains belonging to this genus has been sequenced and the presence of a luxS-like gene was deduced from their genome sequences. The aim of this study was to assess if a complete QS circuit is present in Deinococcus sp. and if this QS is associated with ROS. Primers for searching luxS-like gene and the putative receptor gene, namely ai2R, were designed. AI-2 signal production was evaluated by luminescence analysis using Vibrio harveyi BB170 as reporter strain. AI-2 signal was also evaluated by competitive assays using cinnamaldehyde, ascorbic acid, and 3-mercaptopropionic acid as interfering molecules. Potassium tellurite and metronidazole were used as oxidative stressors. A luxS-like gene as well as an ai2R gene was detected in strain UDEC-P1 by PCR. Cell-free supernatant of strain UDEC-P1 culture induced luminescence in V. harveyi BB170, and this property was inhibited with the three interfering molecules. The oxidative stressors metronidazole and potassium tellurite decreased Deinococcus sp. viability, but increased luminescence of the reporter strain. The results demonstrate that both a functional luxS-like gene and a putative receptor for AI-2 signal are present in Deinococcus sp. strain UDEC-P1. This finding also suggests that a complete QS circuit is present in this genus, which could be related to oxidative stress.     

Antibiofilm activity of sodium bicarbonate, sodium metaperiodate and SDS combination against dental unit waterline-associated bacteria and yeast

Aim: To determine the effect of sodium bicarbonate (SB), sodium metaperiodate (SMP) and sodium dodecyl sulfate (SDS) combination on biofilm formation and dispersal in dental unit waterline (DUWL)-associated bacteria and yeast. Methods and Results: The in vitro effect of SB, SMP and SDS alone and in combination on biofilm formation and dispersal in Pseudomonas aeruginosa, Klebsiella pneumoniae, Actinomyces naeslundii, and Candida albicans was investigated using a 96-well microtitre plate biofilm assay. The combination showed a broad-spectrum inhibitory effect on growth as well as biofilm formation of both gram-negative and gram-positive bacteria, and yeast. In addition, the SB + SMP + SDS combination was significantly more effective in dispersing biofilm than the individual compounds. The combination dispersed more than 90% of P. aeruginosa biofilm whereas the commercial products, Oxygenal 6, Sterilex Ultra, and PeraSafe showed no biofilm dispersal activity. Conclusion: The composition comprising SB, SMP, and SDS was effective in inhibiting as well as dispersing biofilms in DUWL-associated bacteria and yeast. Significance and Impact of the Study: This study shows that a composition comprising environmentally friendly and biologically safe compounds such as SB, SMP, and SDS has a potential application in reducing DUWL-associated acquired infections in dental clinics.     

Chelator-Induced Dispersal and Killing of Pseudomonas aeruginosa Cells in a Biofilm

Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Bacteria in biofilms are more resistant to the immune system and to antibiotics than their free-living planktonic counterparts. Thus, biofilm-related infections are persistent and often show recurrent symptoms. The metal chelator EDTA is known to have activity against biofilms of gram-positive bacteria such as Staphylococcus aureus. EDTA can also kill planktonic cells of Proteobacteria like Pseudomonas aeruginosa. In this study we demonstrate that EDTA is a potent P. aeruginosa biofilm disrupter. In Tris buffer, EDTA treatment of P. aeruginosa biofilms results in 1,000-fold greater killing than treatment with the P. aeruginosa antibiotic gentamicin. Furthermore, a combination of EDTA and gentamicin results in complete killing of biofilm cells. P. aeruginosa biofilms can form structured mushroom-like entities when grown under flow on a glass surface. Time lapse confocal scanning laser microscopy shows that EDTA causes a dispersal of P. aeruginosa cells from biofilms and killing of biofilm cells within the mushroom-like structures. An examination of the influence of several divalent cations on the antibiofilm activity of EDTA indicates that magnesium, calcium, and iron protect P. aeruginosa biofilms against EDTA treatment. Our results are consistent with a mechanism whereby EDTA causes detachment and killing of biofilm cells.     

Investigating the Link Between Imipenem Resistance and Biofilm Formation by Pseudomonas aeruginosa

Pseudomonas aeruginosa, a ubiquitous environmental organism, is a difficult-to-treat opportunistic pathogen due to its broad-spectrum antibiotic resistance and its ability to form biofilms. In this study, we investigate the link between resistance to a clinically important antibiotic, imipenem, and biofilm formation. First, we observed that the laboratory strain P. aeruginosa PAO1 carrying a mutation in the oprD gene, which confers resistance to imipenem, showed a modest reduction in biofilm formation. We also observed an inverse relationship between imipenem resistance and biofilm formation for imipenem-resistant strains selected in vitro, as well as for clinical isolates. We identified two clinical isolates of P. aeruginosa from the sputum of cystic fibrosis patients that formed robust biofilms, but were sensitive to imipenem (MIC?≤?2 μg/ml). To test the hypothesis that there is a general link between imipenem resistance and biofilm formation, we performed transposon mutagenesis of these two clinical strains to identify mutants defective in biofilm formation, and then tested these mutants for imipenem resistance. Analysis of the transposon mutants revealed a role for previously described biofilm factors in these clinical isolates of P. aeruginosa, including mutations in the pilY1, pilX, pilW, algC, and pslI genes, but none of the biofilm-deficient mutants became imipenem resistant (MIC?≥?8 μg/ml), arguing against a general link between biofilm formation and resistance to imipenem. Thus, assessing biofilm formation capabilities of environmental isolates is unlikely to serve as a good predictor of imipenem resistance. We also discuss our findings in light of the limited literature addressing planktonic antibiotic resistance factors that impact biofilm formation.     

Autoinducer-2 Could Affect Biofilm Formation by Food-Derived Bacillus cereus

Bacillus cereus is a foodborne pathogen and cause a frequent problem due to the biofilms forming in equipment of food production plants. Autoinducer-2 (AI-2) involved in interspecies communication, plays a role in the biofilm formation of B. cereus. In this study, biofilm formation by thirty-nine B. cereus strains isolated from foods produced in Korea was determined. To investigate the effect of AI-2 on biofilm formation by B. cereus SBC27, which had the highest biofilm-forming ability, biofilm densities formed after addition of the AI-2 from Staphylococcus aureus and Escherichia coli were analysed. As a result, it was found that the quorum sensing molecule AI-2 could induce biofilm formation by B. cereus within 24 h, but it may also inhibit biofilm formation when more AI-2 is added after 24 h. Thus, these results improve our understanding of biofilm formation by food-derived B. cereus and provide clues that could help to reduce the impact of biofilms, the biggest problem in food processing environments, which has an impact on public health as well as the economy.     

Assessment of the Roles of LuxS, S-Ribosyl Homocysteine, and Autoinducer 2 in Cell Attachment during Biofilm Formation by Listeria monocytogenes EGD-e

LuxS is responsible for the production of autoinducer 2 (AI-2), which is involved in the quorum-sensing response of Vibrio harveyi. AI-2 is found in several other gram-negative and gram-positive bacteria and is therefore considered a good candidate for an interspecies communication signal molecule. In order to determine if this system is functional in the gastrointestinal pathogen Listeria monocytogenes EGD-e, an AI-2 bioassay was performed with culture supernatants. The results indicated that this bacterium produces AI-2 like molecules. A potential ortholog of V. harveyi luxS, lmo1288, was found by performing sequence similarity searches and complementation experiments with Escherichia coli DH5α, a luxS null strain. lmo1288 was found to be a functional luxS ortholog involved in AI-2 synthesis. Indeed, interruption of lmo1288 resulted in loss of the AI-2 signal. Although no significant differences were observed between Lux1 and EGD-e with regard to planktonic growth (at 10°C, 15°C, 25°C, and 42°C), swimming motility, and phospholipase and hemolytic activity, biofilm culture experiments showed that under batch conditions between 25% and 58% more Lux1 cells than EGD-e cells were attached to the surface depending on the incubation time. During biofilm growth in continuous conditions after 48 h of culture, Lux1 biofilms were 17 times denser than EGD-e biofilms. Finally, our results showed that Lux1 accumulates more S-adenosyl homocysteine (SAH) and S-ribosyl homocysteine (SRH) in culture supernatant than the parental strain accumulates and that SRH, but not SAH or AI-2, is able to modify the number of attached cells.     

禽致病性大肠杆菌安徽分离株luxS和pfs基因的克隆、表达与细胞外合成AI-2 活性检测

【目的】LuxS/AI-2型密度感应系统存在于革兰氏阴性和阳性菌中,可产生用于细菌种间交流的通用自诱导信号分子AI-2(Autoinducer-2,AI-2),细菌许多生理功能都受此系统的调节。本研究开展对禽致病性大肠杆菌(Avian Pathogenic Escherichia coli,APEC)自诱导信号分子AI-2的检测和建立体外合成、定量的方法,为进一步研究APEC的AI-2调控作用奠定基础。【方法】利用哈维弧菌BB170(Vibrio harveyi BB170)开展对APEC AI-2的检测;利用表达、纯化的LuxS和Pfs在体外催化S-腺苷同型半胱氨酸(Sadenosylhomocysteine,SAH),进行AI-2的体外合成。【结果】APEC能产生自诱导信号分子AI-2;成功表达可用于AI-2合成的可溶性重组蛋白LuxS和Pfs;纯化的重组蛋白LuxS和Pfs与SAH同时作用后,合成了浓度为300μmol/L的AI-2;运用哈维弧菌BB170对合成的AI-2活性检测表明,其活性是阴性对照的700倍。【结论】APEC存在LuxS/AI-2型密度感应系统,APEC的LuxS和Pfs可以在体外催化SAH生成有活性的AI-2分子。本研究为进一步研究APEC的AI-2的调控作用奠定基础。     

Arctic Actinomycetes as Potential Inhibitors of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Biofilm

The aim of this study was to identify novel biofilm inhibitors from actinomycetes isolated from the Arctic against Vibrio cholerae, the causative agent of cholera. The biofilm inhibitory activity of actinomycetes was assessed using biofilm assay and was confirmed using air–liquid interphase coverslip assay. The potential isolates were identified using 16S rRNA gene sequencing. Of all, three isolates showed significant biofilm inhibition against V. cholerae. The results showed that 20% of the actinomycetes culture supernatant could inhibit up to 80% of the biofilm formation. When different extracted fractions were assessed, significant biofilm inhibition activity was only seen in the diethyl ether fraction of A745. At 200 μg ml⁻¹ of diethyl ether fraction, 60% inhibition of V. cholerae biofilm was observed. The two potential isolates were found to be Streptomyces sp. and one isolate belonged to Nocardiopsis sp. This is the first report showing a Streptomyces sp. and Nocardiopsis sp. isolated from the Arctic having a biofilm inhibitory activity against V. cholerae. The spread of drug resistant V. cholerae strains is a major clinical problem and the ineffectiveness in antibiotic treatment necessitates finding new modes of prevention and containment of the disease, cholera. The formation of biofilms during the proliferation of V. cholerae is linked to its pathogenesis. Hence, the bioactive compound from the culture supernatant of the isolates identified in this study may be a promising source for the development of a potential quorum sensing inhibitors against V. cholerae.