Inhibitory effect of methyl gallate and gallic acid on oral bacteria

This study examined the ability of methyl gallate (MG) and gallic acid (GA), the main compounds of gallo-tannins in Galla Rhois, to inhibit the proliferation of oral bacterial and the in vitro formation of Streptococcus mutans biofilms. The antimicrobial activities of these compounds were evaluated in vitro using the broth microdilution method and a beaker-wire test. Both MG and GA had inhibitory effects on the growth of cariogenic (MIC<8 mg/ml) and periodontopathic bacteria (MIC=1 mg/ml). Moreover, these compounds significantly inhibited the in vitro formation of S. mutans biofilms (MG, 1 mg/ml; GA, 4 mg/ml; P<0.05). MG was more effective in inhibiting bacterial growth and the formation of S. mutans biofilm than GA. In conclusion, MG and GA can inhibit the growth of oral pathogens and S. mutans biofilm formation, and may be used to prevent the formation of oral biofilms.     

Effect of ferrocene-substituted porphyrin RL-91 on Candida albicans biofilm formation

Ferrocene-substituted porphyrin RL-91 exhibits antifungal activity against opportune human pathogen Candida albicans. RL-91 efficiently inhibits growth of both planktonic C. albicans cells and cells within biofilms without photoactivation. The minimal inhibitory concentration for plankton form (PMIC) was established to be 100 μg/mL and the same concentration killed 80% of sessile cells in the mature biofilm (SMIC80). Furthermore PMIC of RL-91 efficiently prevents C. albicans biofilm formation. RL-91 is cytotoxic for human fibroblasts in vitro in concentration of 10 μg/mL, however it does not cause hemolysis in concentrations of up to 50 μg/mL. These findings open possibility for application of RL-91 as an antifungal agent for external antibiofilm treatment of medical devices as well as a scaffold for further development of porphyrin based systemic antifungals.     

Optimized plant compound with potent anti-biofilm activity across gram-negative species

Many human diseases, including cystic fibrosis lung infections, are caused or exacerbated by bacterial biofilms. Specialized modes of motility, including swarming and twitching, allow gram-negative bacteria to spread across surfaces and form biofilms. Compounds that inhibit these motilities could slow the spread of biofilms, thereby allowing antibiotics to work better. We previously demonstrated that a set of plant-derived triterpenes, including oleanolic acid and ursolic acid, inhibit formation of Escherichia coli and Pseudomonas aeruginosa biofilms, and alter expression of genes involved in chemotaxis and motility. In the present study, we have prepared a series of analogs of oleanolic acid. The analogs were evaluated against clinical isolates of E. coli and P. aeruginosa in biofilm formation assays and swarming assays. From these analogs, compound 9 was selected as a lead compound for further development. Compound 9 inhibits E. coli biofilm formation at 4 µg/mL; it also inhibits swarming at ≤1 µg/mL across multiple clinical isolates of P. aeruginosa, E. coli, Burkholderia cepacia, and Salmonella enterica, and at <0.5 µg/mL against multiple agricultural strains. Compound 9 also potentiates the activity of the antibiotics tobramycin and colistin against swarming P. aeruginosa; this is notable, as tobramycin and colistin are inhaled antibiotics commonly used to treat P. aeruginosa lung infections in people with cystic fibrosis. qPCR experiments suggested that 9 alters expression of genes involved in regulating Type IV pili; western blots confirmed that expression of Type IV pili components PilA and PilY1 decreases in P. aeruginosa in the presence of 9.     

Identification of aryl 2-aminoimidazoles as biofilm inhibitors in Gram-negative bacteria

The synthesis and biofilm inhibitory activity of a 30-member aryl amide 2-aminoimidazole library against the three biofilm forming Gram-negative bacteria Escherichia coli, Psuedomonas aeruginosa, and Acinetobacter baumannii is presented. The most active compound identified inhibits the formation of E. coli biofilms with an IC₅₀ of 5.2 μM and was observed to be non-toxic to planktonic growth, demonstrating that analogues based on an aryl framework are viable options as biofilm inhibitors within the 2-aminoimidazole family.     

The human antimicrobial peptide LL-37 and its fragments possess both antimicrobial and antibiofilm activities against multidrug-resistant Acinetobacter baumannii

Acinetobacter baumannii infections are difficult to treat due to multidrug resistance. Biofilm formation by A. baumannii is an additional factor in its ability to resist antimicrobial therapy. The antibacterial and antibiofilm activities of the human antimicrobial peptide LL-37 and its fragments KS-30, KR-20 and KR-12 against clinical isolates of multidrug-resistant (MDR) A. baumannii were evaluated. The minimal inhibitory concentration (MIC) of LL-37 against MDR A. baumannii isolates ranged from 16 to 32 μg/mL. The MIC of KS-30 fragment varied from 8.0 to16 μg/mL and the KR-20 fragment MIC ranged from 16 to 64 μg/mL. LL-37 and KS-30 fragment exhibited 100% bactericidal activity against five A. baumannii strains, including four MDR clinical isolates, within 30 min at concentrations of 0.25–1 μg/mL. By 0.5 h, the fragments KR-20 and KR-12 eliminated all tested strains at 8 and 64 μg/mL respectively. LL-37 and its fragments displayed anti-adherence activities between 32-128 μg/mL. A minimum biofilm eradication concentration (MBEC) biofilm assay demonstrated that LL-37 inhibited and dispersed A. baumannii biofilms at 32 μg/mL respectively. Truncated fragments of LL-37 inhibited biofilms at concentrations of 64–128 μg/mL. KS-30, the truncated variant of LL-37, effectively dispersed biofilms at 64 μg/mL. At 24 h, no detectable toxicity was observed at the efficacious doses when cytotoxicity assays were performed. Thus, LL-37, KS-30 and KR-20 exhibit significant antimicrobial activity against MDR A. baumannii. The prevention of biofilm formation in vitro by LL-37, KS-30 and KR-20 adds significance to their efficacy. These peptides can be potential therapeutics against MDR A. baumannii infections.     

白花丹素对E.coli藻酸盐合成的抑制作用

细菌分泌的胞外多糖在生物被膜的形成和发展过程中发挥着重要作用。通过测定白花丹素对大肠埃希菌10389菌株(E.coli 10389)藻酸盐合成的影响及其对rse A和rpo E基因表达量的影响,探讨白花丹素对大肠埃希菌生物被膜(biofilm,BF)形成的抑制作用及机制。研究结果显示,白花丹素能抑制E.coli 10389生物被膜的形成,其抑杀E.coli 10389的最低抑菌浓度(minimum inhibitory concentration,MIC)和最低杀菌浓度(minimal bactericidal concentration,MBC)分别为16和64μg/mL。白花丹素对成熟BF内的细菌也有抑制和杀灭作用,其抑杀E.coli 10389成熟BF内细菌的MIC和MBC分别为64和128μg/mL。白花丹素能够抑制E.coli 10389藻酸盐的合成,其中1/2MIC的白花丹素作用E.coli 10389 24 h后,与对照组比,藻酸盐的合成量降低了34.83%(P0.01)。白花丹素可显著影响E.coli 10389 rse A和rpo E基因的相对表达量,其中1/2MIC的白花丹素作用E.coli 10389 24 h后,与对照组相比,rse A的表达量上调了17.43%,rpo E的表达量降低了12.8%(P0.05)。结果表明,白花丹素能够抑制E.coli 10389 BF的形成,其作用机制可通过影响rse A和rpo E的基因表达量,进而抑制藻酸盐的合成来抑制大肠埃希菌生物被膜的形成。     

Biofilm formation as a novel phenotypic feature of adherent-invasive Escherichia coli (AIEC)

Background  

Crohn's disease (CD) is a high morbidity chronic inflammatory disorder of unknown aetiology. Adherent-invasive Escherichia coli (AIEC) has been recently implicated in the origin and perpetuation of CD. Because bacterial biofilms in the gut mucosa are suspected to play a role in CD and biofilm formation is a feature of certain pathogenic E. coli strains, we compared the biofilm formation capacity of 27 AIEC and 38 non-AIEC strains isolated from the intestinal mucosa. Biofilm formation capacity was then contrasted with the AIEC phenotype, the serotype, the phylotype, and the presence of virulence genes.     

Fimbria targeting superparamagnetic iron oxide nanoparticles enhance the antimicrobial and antibiofilm activity of ciprofloxacin against quinolone-resistant E. coli

High quinolone resistance of Escherichia coli limits the therapy options for urinary tract infection (UTI). In response to the urgent need for efficient treatment of multidrug-resistant infections, we designed a fimbriae targeting superparamagnetic iron oxide nanoparticle (SPION) delivering ciprofloxacin to ciprofloxacin-resistant E. coli. Bovine serum albumin (BSA) conjugated poly(acrylic acid) (PAA) coated SPIONs (BSA@PAA@SPION) were developed for encapsulation of ciprofloxacin and the nanoparticles were tagged with 4-aminophenyl-α-D-mannopyrannoside (mannoside, Man) to target E. coli fimbriae. Ciprofloxacin-loaded mannoside tagged nanoparticles (Cip-Man-BSA@PAA@SPION) provided high antibacterial activity (97.1 and 97.5%, respectively) with a dose of 32 μg/mL ciprofloxacin against two ciprofloxacin-resistant E. coli isolates. Furthermore, a strong biofilm inhibition (86.9% and 98.5%, respectively) was achieved in the isolates at a dose 16 and 8 times lower than the minimum biofilm eradication concentration (MBEC) of ciprofloxacin. Weaker growth inhibition was observed with untargeted nanoparticles, Cip-BSA@PAA@SPIONs, confirming that targeting E. coli fimbria with mannoside-tagged nanoparticles increases the ciprofloxacin efficiency to treat ciprofloxacin-resistant E. coli. Enhanced killing activity against ciprofloxacin-resistant E. coli planktonic cells and strong growth inhibition of their biofilms suggest that Cip-Man-BSA@PAA@SPION system might be an alternative and/or complementary therapeutic option for the treatment of quinolone-resistant E. coli infections.     

Modulation of <Emphasis Type="Italic">Escherichia coli</Emphasis> biofilm growth by cell-free spent cultures from lactobacilli

E. coli biofilms cause serious problems in medical practice by contaminating surfaces and indwelling catheters. Due to the rapid development of antibiotic resistance, alternative approaches to biofilm suppression are needed. This study addresses whether products released by antagonistic bacteria — Lactobacillus isolates from vaginal and dairy-product samples could be useful for controlling E. coli biofilms. The effects of diluted cell-free supernatants (CFS) from late-exponential Lactobacillus cultures on the growth and biofilm production of Escherichia coli were tested. Most of the CFS applied as 10⁻² had no impact on bacterial growth, biofilm development however was influenced even by 10⁻⁴ of CFS. Initial screening by crystal violet assay showed that biofilm modulation varied between different CFS and E. coli combinations from inhibition to activation; however three of the tested CFS showed consistency in biofilm suppression. This was not due to antibacterial activity since Live/Dead fluorescence labeling showed insignificant differences in the amount of dead cells in control and treated samples. Some E. coli strain-specific mechanisms of response to the three CFS included reduction in hydrophobicity and motility. Released exoploysaccharides isolated from the three CFS stimulated sessile growth, but proteinase K reduced their inhibitory activities implying participation of protein or peptide biofilm suppression factor(s).     

The radish defensins RsAFP1 and RsAFP2 act synergistically with caspofungin against Candida albicans biofilms

The radish defensin RsAFP2 was previously characterized as a peptide with potent antifungal activity against several plant pathogenic fungi and human pathogens, including Candida albicans. RsAFP2 induces apoptosis and impairs the yeast-to-hypha transition in C. albicans. As the yeast-to-hypha transition is considered important for progression to mature biofilms, we analyzed the potential antibiofilm activity of recombinant (r)RsAFP2, heterologously expressed in Pichia pastoris, against C. albicans biofilms. We found that rRsAFP2 prevents C. albicans biofilm formation with a BIC-2 (i.e., the minimal rRsAFP2 concentration that inhibits biofilm formation by 50% as compared to control treatment) of 1.65 ± 0.40 mg/mL. Moreover, biofilm-specific synergistic effects were observed between rRsAFP2 doses as low as 2.5 μg/mL to 10 μg/mL and the antimycotics caspofungin and amphotericin B, pointing to the potential of RsAFP2 as a novel antibiofilm compound. In addition, we characterized the solution structure of rRsAFP2 and compared it to that of RsAFP1, another defensin present in radish seeds. These peptides have similar amino acid sequences, except for two amino acids, but rRsAFP2 is more potent than RsAFP1 against planktonic and biofilm cultures. Interestingly, as in case of rRsAFP2, also RsAFP1 acts synergistically with caspofungin against C. albicans biofilms in a comparable low dose range as rRsAFP2. A structural comparison of both defensins via NMR analysis revealed that also rRsAFP2 adopts the typical cysteine-stabilized αβ-motif of plant defensins, however, no structural differences were found between these peptides that might result in their differential antifungal/antibiofilm potency. This further suggests that the conserved structure of RsAFP1 and rRsAFP2 bears the potential to synergize with antimycotics against C. albicans biofilms.     

Synthesis of benzoylthiourea derivatives and analysis of their antibacterial performance against planktonic Staphylococcus aureus and its biofilms

Following the appearance of several antimicrobial agents to control the spread of infections, two major challenges have emerged: (i) the occurrence and blowout of multiresistant bacteria and the increase of chronic diseases and (ii) difficult-to-eradicate infections. In this study, we tested five benzoylthiourea derivatives for their ability to inhibit and stop bacterial growth and evaluated the possible influence of 1,2,4-triazolyl-benzoylthiourea derivative 4 on the formation and eradication of Staphylococcus aureus biofilms. Benzoylthiourea derivatives 4 , 6 , 10 , 11 and 13 were obtained in one or two steps with low cost and subjected to tests to identify their minimum inhibitory concentration (MIC) and minimum bactericidal concentration. In vitro tests were also performed to assess their effects on biofilm formation and in preformed biofilms and scanning electron microscopy was used to visualize the effects on biofilm formation. The 1,2,4-triazolyl-benzoylthiourea derivative 4 showed bacteriostatic activity against the S. aureus HU25 clinical strain with an MIC of 16 µg ml⁻¹, which is below the toxic concentration (at 2500 µg ml⁻¹, 62·25% of the cells remained viable). Compound 4 also effectively prevented biofilm formation at the three subinhibitory concentrations tested (1/2 MIC, 1/4 MIC and 1/8 MIC) as confirmed by scanning electron microscopy. For breakdown of formed biofilms, the main influence was at a subinhibitory concentration (1/2 MIC). These findings make compound 4 a strong candidate for studies on the development of new antimicrobial and antibiofilm agents.     

壳聚糖-纳米羟基磷灰石复合制剂对粪肠球菌抗菌作用的体外研究

目的观察壳聚糖一纳米羟基磷灰石（CS／nHA）复合制剂对粪肠球菌生物膜的体外抗菌作用,为研究该药物对根管内粪肠球菌生物膜抗菌的作用打下基础,探索激光共聚焦显微镜（CLSM）技术在研究生物膜领域中的优势。方法采用液体二倍稀释法,测定壳聚糖／纳米羟基磷灰石复合制剂对游离状态下粪肠球菌的最小抑菌浓度和最小杀菌浓度;将粪肠球菌在体外培养成生物膜后,利用CLSM观察研究该生物膜中细菌活性。结果CS／nHA制剂在0．467mg／mL时对游离状态下的粪肠球菌具有抑菌和杀菌作用,与对照组相比差异有统计学意义。使用CS／nHA制剂前后粪肠球菌生物膜活菌百分比显著下降,与对照组相比差异有统计学意义（P〈0．05）。结论CS／nHA制剂对粪肠球菌有较强的抗菌作用,将其作为根管封闭剂中的抗菌成分具有一定的应用前景。     

Inhibition and destruction of Pseudomonas aeruginosa biofilms by antibiotics and antimicrobial peptides

Pseudomonas aeruginosa is one of the major nosocomial pathogen that can causes a wide variety of acute and chronic infections P. aeruginosa is a dreaded bacteria not just because of the high intrinsic and acquired antibiotic resistance rates but also the biofilm formation and production of multiple virulence factors. We investigated the in vitro activities of antibiotics (ceftazidime, tobramycin, ciprofloxacin, doripenem, piperacillin and colistin) and antimicrobial cationic peptides (AMPs; LL-37, CAMA: cecropin(1–7)-melittin A(2–9) amide, melittin, defensin and magainin-II) alone or in combination against biofilms of laboratory strain ATCC 27853 and 4 clinical strains of P. aeruginosa. The minimum inhibitory concentrations (MIC), minimum bactericidal concentration (MBC) and minimum biofilm eradication concentrations (MBEC) were determined by microbroth dilution technique. The MBEC values of antibiotics and AMPs were 80–>5120 and 640–>640 mg/L, respectively. When combined with the LL-37 or CAMA at 1/10× MBEC, the MBEC values of antibiotics that active against biofilms, were decreased up to 8-fold. All of the antibiotics, and AMPs were able to inhibit the attachment of bacteria at the 1/10× MIC and biofilm formation at 1× or 1/10× MIC concentrations. Time killing curve studies showed 3-log₁₀ killing against biofilms in 24 h with almost all studied antibiotics and AMPs. Synergism were seen in most of the studied combinations especially CAMA/LL-37 + ciprofloxacin against at least one or two strains’ biofilms. Since biofilms are not affected the antibiotics at therapeutic concentrations, using a combination of antimicrobial agents including AMPs, or inhibition of biofilm formation by blocking the attachment of bacteria to surfaces might be alternative methods to fight with biofilm associated infections.     

Biofilm Formation Protects Escherichia coli against Killing by Caenorhabditis elegans and Myxococcus xanthus

Enteric bacteria, such as Escherichia coli, are exposed to a variety of stresses in the nonhost environment. The development of biofilms provides E. coli with resistance to environmental insults, such as desiccation and bleach. We found that biofilm formation, specifically production of the matrix components curli and cellulose, protected E. coli against killing by the soil-dwelling nematode Caenorhabditis elegans and the predatory bacterium Myxococcus xanthus. Additionally, matrix-encased bacteria at the air-biofilm interface exhibited ∼40-fold-increased survival after C. elegans and M. xanthus killing compared to the non-matrix-encased cells that populate the interior of the biofilm. To determine if nonhost Enterobacteriaceae reservoirs supported biofilm formation, we grew E. coli on media composed of pig dung or commonly contaminated foods, such as beef, chicken, and spinach. Each of these medium types provided a nutritional environment that supported matrix production and biofilm formation. Altogether, we showed that common, nonhost reservoirs of E. coli supported the formation of biofilms that subsequently protected E. coli against predation.     

Convenient framework of poly functionalized (E)-2-benzylideno-(Z)-carbazolylideno cyanoacetamides via rearrangements as an efficient antibiofilm inhibitors with SAR study

A simple and one-pot approach for the synthesis of highly functionalized novel (E)-2-benzylideno-(Z)-carbazolylideno cyanoacetamide derivatives from different 2-(2′,3′,4′,9′-tetrahydro-carbazol-1′-ylidene)-propanedinitriles and aryl/heteroaryl carbaldehydes via vinylogous aldol reaction. The structures of the molecules were designated by FT-IR, ¹H NMR, ¹³C NMR studies, elemental and X-ray crystallographic analysis. The synthesized pure products have been screened for in vitro antibiofilm inhibitory activity towards antibiotic-resistant pathogenic organisms. All the synthesized compounds showed biofilm inhibition. Promisingly, the moieties 3a, 3d and 3h showed higher antibiofilm activity at biofilm inhibitory concentration (BIC) (200?μg/mL) against bacterial pathogens. Among the three moieties, 3a showed high prospective against E. coli biofilm with minimal and maximal BIC percentage of 32% (10?μg/mL) and 89% (100?μg/mL) and chosen lowest BIC for further evaluation. Also, the 3a generate ROS two fold at 1?h treatment in E. coli biofilm. The 3a exhibited no toxic effect on cell viability upto 75?μg/mL in HEK293 cell lines. The results of the present study reveal that among (E)-2-benzylideno-(Z)-carbazolylideno cyanoacetamides, (E)-2-benzylideno-6-methyl-2,3,4,9-tetrahydro-1H-carbazol-(Z)-α-carbamino-α-cyano-1-ylidene (3a) could be exploited as an excellent antibiofilm agent against carbapenem-resistant E. coli bacteria strains.     

Interaction between atypical microorganisms and E. coli in catheter-associated urinary tract biofilms

Most biofilms involved in catheter-associated urinary tract infections (CAUTIs) are polymicrobial, with disease causing (eg Escherichia coli) and atypical microorganisms (eg Delftia tsuruhatensis) frequently inhabiting the same catheter. Nevertheless, there is a lack of knowledge about the role of atypical microorganisms. Here, single and dual-species biofilms consisting of E. coli and atypical bacteria (D. tsuruhatensis and Achromobacter xylosoxidans), were evaluated. All species were good biofilm producers (Log 5.84–7.25 CFU cm^?2 at 192?h) in artificial urine. The ability of atypical species to form a biofilm appeared to be hampered by the presence of E. coli. Additionally, when E. coli was added to a pre-formed biofilm of the atypical species, it seemed to take advantage of the first colonizers to accelerate adhesion, even when added at lower concentrations. The results suggest a greater ability of E. coli to form biofilms in conditions mimicking the CAUTIs, whatever the pre-existing microbiota and the inoculum concentration.     

Inhibition of Streptococcus pyogenes Biofilm Formation by Coral-Associated Actinomycetes

Streptococcus pyogenes biofilms tend to exhibit significant tolerance to antimicrobials during infections. We screened coral-associated actinomycetes (CAA) for antibiofilm activity against different biofilm forming M serotype of Streptococcus pyogenes. Actinomycetes isolated from the mucus of the coral Acropora digitifera were screened for antibiofilm activity against S. pyogenes biofilms wherein several isolates clearly demonstrated antibiofilm activity. The biofilm inhibitory concentrations (BICs) and the sub-BICs (1/2 and 1/4 BIC) of the extracts significantly prevented biofilm formation up to 60–80%. The extract of Streptomyces akiyoshinensis (A3) displayed efficient antibiofilm activity against all the biofilm forming M serotypes. All the five extracts efficiently reduced the cell surface hydrophobicity (a crucial factor for biofilm formation in S. pyogenes) of three M types and thus may inhibit biofilm formation. CAA represent an interesting source of marine invertebrates-derived antibiofilm agents in the development of new strategies to combat Streptococcal biofilms.