Biofilm Inhibition Assay Research Articles

Quantification of anti-quorum sensing and antibiofilm activity of Phyllosphere bacteria against food spoilage bacteria

Food spoilage and microbial contamination require attention during the food production process since the presence of these bacteria can create problems including the formation of biofilms produced by these bacteria. Biofilm formations are initiated through cell-to-cell communication which is called quorum sensing mechanism. Hence, inhibition of this communication mechanism could be one of the solutions to inhibit biofilm formation. Therefore, exploration of bioactive compounds from various sources including hyllosphere bacteria with anti-quorum sensing inhibition activities is important. Phyllosphere bacteria are a community of bacteria found on the surface of plant leaves at a very large population. These bacteria can produce bioactive compounds that can inhibit quorum sensing mechanism. In this study, 54 phyllosphere bacteria isolates were tested, 8 bacterial isolates had potential effect to inhibit quorum sensing. From biofilm inhibition assay, the highest percentages were showed by ifferent phyllosphere isolates against each pathogen. Whereas, for biofilm destruction assay, JB 8F isolate had the highest percentage of destruction biofilm activity against biofilm formed by Bacillus cereus and Shewanella putrefaciens. Eight isolates of phyllosphere bacteria had the potential as quorum quencher and anti-biofilm agents, both for inhibition and destruction of biofilm.

UPLC-ESI-MS/MS profiling of the underground parts of common Iris species in relation to their anti-virulence activities against Staphylococcusaureus

Ethnopharmacological relevanceThe use of plant extracts and their phytochemicals as candidates for targeting the microbial resistance inhibition is increasingly focused in last decades. In Mongolian traditional medicine, Irises were long used for the treatment of bacterial infections. Irises have been used since the Ancient Egyptians. Aim of the studyChemical composition and virulence inhibition potential of both polar (PF) and non-polar fractions (NPF) of three common Iris species (I. confusa, I. pseudacorus and I. germanica) were explored. Material and methodsSecondary metabolites profiling was characterized by the UPLC-HRMS/MS technique. Multi-variate data analysis was performed using Metaboanalyst 3.0. Anti-virulence inhibitory activity was evaluated via anti-haemolytic assay and Quantitative biofilm inhibition assay. ResultsI. pseudacorus PF exhibited the most potent effect against S. aureus haemolytic activity. All the tested fractions from all species, except I. pseudacorus NPF, have no significant inhibition on the biofilm formation of methicillin resistant and sensitive (MRSA and MSSA) S. aureus. I. pseudacorus NPF showed potent biofilm inhibitory potential of 71.4 and 85.8% against biofilm formation of MRSA and MSSA, respectively. Metabolite profiling of the investigated species revealed ninety and forty-five metabolites detected in the PFs and NPFs, respectively. Nigricin-type, tectorigenin-type isoflavonids and xanthones allowed the discrimination of I. pseudacorus PF from the other species, highlighting the importance of those metabolites in exerting its promising activity. On the other hand, triterpene acids, iridals, triacylglycerols and ceramides represented the metabolites detected in highest abundance in I. pseudacorus NPF. ConclusionsThis is the sole map represents the secondary metabolites profiling of the PFs and NPFs of common Iris species correlating them with the potent explored Staphylococcus aureus anti-virulence activity.

Development of SphagneticolaTrilobata Flowers Extract Coated Urinary Catheters: Determining Its Antibacterial Activity against Escherichia coli and Staphylococcus aureus

Urinary catheters are regularly used withinside the elderly for each short- or long-term. The biofilm formation occurs on the catheters and leads to urinary tract infection. The present study focus on the development of Sphagneticolatrilobata coated catheters for the prevention of urinary tract infections (UTI). Bioactive compounds were extracted using methanol and MIC was determined. Biofilm inhibition assay was determined by Fluorescent microscopic analysis. 7.5mg/ml extracts showed 13mm against E. coli and 14mm against S. aureus. The extract-coated catheter showed a significant reduction in biofilms from the fluorescent microscopic analysis. Hence Sphagneticolatrilobataflower extract coated catheters can be used to prevent biofilm colonization and urinary tract infections.

Enhanced anti-biofilm activity of facile synthesized silver oxide nanoparticles against K. pneumoniae

The silver oxide nanoparticle was successfully synthesized using floral waste by simple one pot, cost effective method. The complete formation of silver oxide nanoparticles was characterized by UV-spec, FT-IR, powder XRD. The morphological structure of synthesized Ag2O NPs was confirmation by FE-SEM. The biofilm inhibition and biofilm metabolic assay results were confirmed that the synthesized Ag2O NPs very effective against biofilm formation. In addition, exopolysaccharide and hydrophobicity degradation assays were supported to biofilm inhibition at 250 µg/mL concentration. In addition, the architectural and extracellular damages of K. pneumoniae were proved by CLSM and SEM. Finally, the reported hemolytic activity effect of synthesized Ag2O NPs shown at very low compared with CLSI guidelines, this might be due to the presence of the polyphenols of floral waste extract. Conclusively, the synthesized Ag2O NPs was eco-friendly nature and promising anti-biofilm agent against K. pneumoniae

Synthesis of β-carboline fatty alcohol hybrid molecules and characterization of their biological and antioxidant activities

Nine new β-carboline fatty alcohol hybrids were synthesized from natural amino acid L-tryptophan by the conjugation of 10-undecen-1-ol via ester linkage. All the synthesized products were characterized in each step by spectral techniques (1H, 13C NMR mass and HRMS). Synthesized β-carboline derivatives (7a-i) were screened for biological activities such as antimicrobial, antifungal, anti-biofilm and anticancer. Compounds 7d and 7f showed most potent antimicrobial activity against Bacillus subtilis MTCC 121, Micrococcus luteus MTCC 2470, Staphylococcus aureus MTCC 96 and Candida albicans MTCC 3017 microbial strains ranged from 2.8 to 28.3 µg/mL. Biofilm inhibition assay showed that the compound 7f exhibited better activity against three bacterial stains with IC50 values from 1.8 to 2.9 µM. All the β-carboline derivatives showed cytotoxic activity, among them compounds 7f and 7h (IC50 values 9.1 and 11.4 µM) were exhibited potential activity. Free radical scavenging activity via DPPH assay revealed that compound 7g acted as a good antioxidant molecule than all the tested compounds.

Effect of lemongrass essential oil against multidrug-resistant Salmonella Heidelberg and its attachment to chicken skin and meat

Salmonella Heidelberg (S. Heidelberg) is a major pathogen implicated in foodborne outbreaks for which poultry products can serve as an epidemiological source. This study determined the efficacy of GRAS-status lemongrass essential oil (LGEO) against S. Heidelberg in vitro and on the pathogen's attachment to skin and meat. At first, employing in vitro assays, the effect of LGEO on multidrug-resistant S. Heidelberg multiplication and motility was examined. Biofilm inhibition and inactivation assays were also performed. The quorum-sensing modulating effect of LGEO was determined. In follow-up experiments, chicken skin or meat samples inoculated with S. Heidelberg were treated with various concentrations of LGEO at different time points at simulated scalding (54°C) and chilling (4°C) temperatures. The samples were incubated, and the surviving populations of S. Heidelberg were enumerated to determine if LGEO could be a potential processing aid in poultry operations. Duplicate samples were included in each treatment, and the experiments were repeated at least 3 times. Significant reductions of S. Heidelberg of at least 4.0 log10 CFU/mL after 24 h in nutrient broth and poultry cecal contents was observed with 0.5% LGEO. Complete inhibition of motility, biofilm formation, and inactivation of pre-formed biofilms was observed with 0.15% LGEO (P ≤ 0.05). Concentrations of LGEO at 0.5% and 1% affected violacein production (P ≤ 0.05). On skin samples, all concentrations significantly reduced S. Heidelberg by 1.2 to 3.9 log10 CFU/sample after 2 min at 54°C. We obtained a significant reduction of the pathogen in meat samples at 54°C and skin samples at 4°C with 2% LGEO. All concentrations significantly reduced S. Heidelberg from the treatment water kept at 4°C and 54°C (P ≤ 0.05). In conclusion, LGEO could potentially serve as a natural antimicrobial strategy in scalding and chilling waters to reduce S. Heidelberg during processing. However, additional studies are warranted before recommending its commercial use.

Effects of Essential Oils from Cymbopogon spp. and Cinnamomum verum on Biofilm and Virulence Properties of Escherichia coli O157:H7.

Every year, the pharmaceutical and food industries produce over 1000 tons of essential oils (EOs) exploitable in different fields as the development of eco-friendly and safe antimicrobial inhibitors. In this work we investigated the potential of some EOs, namely Cinnamomum verum, Cymbopogon martini, Cymbopogon citratus and Cymbopogon flexuosus, on the growth, biofilm formation and gene expression in four strains of enterohemorrhagic Escherichia coli O157:H7. All EOs were analyzed by gas chromatography-mass spectrometry (GC-MS). The antimicrobial activity was performed by using dilutions of EOs ranging from 0.001 to 1.2% (v/v). Subinhibitory doses were used for biofilm inhibition assay. The expression profiles were obtained by RT-PCR. E. coli O157:H7 virulence was evaluated in vivo in the nematode Caenorhabditis elegans. All EOs showed minimal inhibitory concentrations (MICs) ranging from 0.0075 to 0.3% (v/v). Cinnamomum verum bark EO had the best activity (MIC of 0.0075% (v/v) in all strains) while the C. verum leaf EO had an intermediate efficacy with MIC of 0.175% (v/v) in almost all strains. The Cymbopogon spp. showed the more variable MICs (ranging from 0.075 to 0.3% (v/v)) depending on the strain used. Transcriptional analysis showed that C. martini EO repressed several genes involved in biofilm formation, virulence, zinc homeostasis and encoding some membrane proteins. All EOs affected zinc homeostasis, reducing ykgM and zinT expression, and reduced the ability of E. coli O157:H7 to infect the nematode C. elegans. In conclusion, we demonstrated that these EOs, affecting E. coli O157:H7 infectivity, have a great potential to be used against infections caused by microorganisms.

ANTI-BIOFILM POTENTIAL OF MENTHOL PURIFIED FROM MENTHA PIPERITA L. (MINT)

Menthol, a bioactive compound of Mentha piperita (mint) with antibacterial properties was purified by column chromatography to determine its anti-biofilm potential. After phytochemical analysis, TLC was carried out using n-hexane: ethyl acetate: methanol: water (2:2:2:1) as the solvent system for ethanolic extract of mint. TLC achieved the maximum separation of mint constituents with Rf value of 0.68. A purified menthol fraction was obtained after silica gel column chromatography using four different eluting solvents. The menthol obtained was then used to perform biofilm inhibition assay to establish its antibacterial potential. Percentage inhibition was highest for bacillus subtilis (79.4%), as opposed to Pseudomonas aeruginosa (33.6%) and the combination of both bacteria (20%). ELISA reader was used to measure absorbance at 450-620nm and 630 nm. Using 450-620nm filter the values for percentage inhibition lies between 48.6-95% for standard and crude menthol samples. Similarly, at 630nm the values of inhibition lie between 23.4-70.6%. This anti-biofilm property of menthol can be utilized in antibacterial drug formulations.

Myco-nanocolloids manipulate growth, biofilm formation and virulence genes in UTI causing E. coli

The existing antibiotics are incapable of killing resistant pathogens including E. coli. Extended-spectrum beta-lactamases (ESBL) genes are found as one of the main causes of the resistance as it degrades the β- lactam ring of the antibiotics. This study aims at the green synthesis of silver nanocolloids from endophytic fungi Lasiodiplodia theobromae (LtNc’s), isolated from traditional medicinal plant Cinnamomum zeylanicum and biophysically characterized. Minimum susceptibility concentration, growth rate, Minimum Bactericidal Concentration and Biofilm inhibition assays were performed to evaluate the efficacy of LtNc’s against biofilm forming urinary tract infection (UTI) causing clinical strains of E. coli. The LtNc’s was found to be efficient antimicrobial agent which successfully controlled the growth and biofilm formation in E. coli. Inhibition of growth, biofilm formation, downregulation of CTX-M-15 and Amp C genes were observed when treated with LtNc’s in Urinary Tract infection (UTI) causing pathogens.

Therapeutic Potential of Selected Varieties of Phoenix Dactylifera L. Against Microbial Biofilm and Free Radical Damage to DNA.

Phoenix dactylifera L. (Date palm) is the most widely consumed fruit around the world and is rich source of nutrients containing dietary fibers, minerals, vitamins, sugar, protein and antioxidants with potent bioactivities against various microbial pathogens. This study evaluated the therapeutic potential of 2 varieties of ethanolic extracts of Phoenix dactylifera i-e Ajwa and Khalas against bacterial biofilms. This study also investigated the protective effect of Ajwa and Khalas against hydroxyl radical damage to calf thymus DNA. Antioxidant potential through different antioxidant assays showed that Ajwa has higher antioxidant potential than Khalas. Both Ajwa and Khalas presented good antimicrobial activities against Bacillus subtilis and Pasteurella multocida. Biofilm inhibition assay showed that increasing concentration of Ajwa and Khalas exhibited higher percentage of bacterial biofilm inhibition. Microscopic examination revealed significant inhibition of microbial biofilm. Ajwa and Khalas protected the calf thymus DNA against damage caused by hydroxyl radicals produced by fenton reagent. Fourier Transform Infrared (FTIR) spectra confirmed the presence of O–H, C=C and C–O functional groups in tested extracts. The study concluded that tested varieties of Date palm have the potential to inhibit bacterial biofilms and can be used for therapeutic purposes against biofilm producing pathogens.

Fabrication and antibacterial activity of nanoenhanced conjugate of silver (I) oxide with graphene oxide

Due to the globally identified antibiotic resistance among clinical bacteria, novel antibacterial materials are needed to circumvent drug resistance. In this study, we report a facile synthesis method for the fabrication of a uniform silver (I) oxide (Ag2O) garnished graphene oxide (GO) nanocomposite (Ag2O/GO) using sonication and characterized them by X-ray diffraction (XRD), Raman spectroscopy, and UV–vis spectroscopy. Furthermore, the antibacterial properties of Ag2O, GO, and Ag2O/GO nanocomposite were studied using drug resistant Gram-negative Escherichia coli (ESBL), Pseudomonas aeruginosa (ESBL), Klebsiella pneumoniae and Gram-positive Staphylococcus aureus by well diffusion assay, colony forming ability, and cell membrane permeability assay. The nanocomposite (15.62−1000 μg/mL) showed excellent antibacterial activity with minimum inhibitory concentration of 125 μg/mL for P. aeruginosa and K. pneumoniae; 62.5 μg/mL for E. coli, and 250 μg/mL for S. aureus. Biofilm inhibition assay revealed the inhibition of biofilm formation in a dose related manner. Inhibition of biofilm formation by ½ MIC and MIC of Ag2O and Ag2O/GO against four pathogenic and biofilm forming bacteria was found statistically significant as P ≤ 0.05 (at ½ MIC) and P ≤ 0.01 (at MIC). These results demonstrated that these facile nanomaterials are promising antibacterial and antibiofilm agents that can be utilized to inhibit the growth of human bacterial pathogens associated with chronic infections.

In-vitro Investigation of Antibiotics Efficacy Against Uropathogenic Escherichia coli Biofilms and Antibiotic Induced Biofilm Formation at Sub-Minimum Inhibitory Concentration of Ciprofloxacin.

BackgroundCommunity-acquired urinary tract infections are associated with significant morbidity, and uropathogenic Escherichia coli (UPEC) alone causes 90% of urinary tract infections. This bacterium retains a diverse armament of virulence factors including fimbria, hemolysins, and siderophores production. In a post invasion scenario, formation of intracellular communities mimic biofilm-like characteristics and are linked to recurrent urinary tract infections. We investigated the effects of different frontline antibiotics on the formation, inhibition, and eradication of biofilms of virulent UPEC strains.Materials and MethodsA total of 155 UPEC strains were scrutinized for various virulence factors including gelatinase, cell surface hydrophobicity, hemagglutination, and serum bactericidal activity. Biofilm formation was confirmed by three different methods: Congo red agar, test tube, and tissue culture plate method. Biofilm inhibition and eradication assays were performed according to the standard protocols. Topographical analysis of biofilms was done by scanning electronic microscopy (SEM).ResultsOut of 155 strains, 113 (73%) were strong biofilm formesr, while 37 (24%) produced biofilms at moderate level. Significant differences were observed between MICs of planktonic cells (MIC-p) and MICs of UPEC biofilms (MIC-b). Among tested frontline antibiotics, levofloxacin successfully inhibited biofilms at a concentration of 32 µg/mL, while trimethoprim eradicated biofilms at higher concentrations (512–1024 µg/mL). Ciprofloxacin treatment at sub-MIC level significantly enhanced biofilm formation (P<0.05).ConclusionThe majority of UPEC strains are strong biofilm formers and show higher tolerance towards frontline antibiotics in biofilm form. We observed significant inhibitory effects of levofloxacin (32 µg/mL) on UPEC biofilms, while treatment with sub-minimal concentrations of ciprofloxacin significantly enhanced biofilm formation. Out of all tested antibiotics, trimethoprim (512–1024 µg/mL) eradicated UPEC biofilms.

Biopolymer Coating of Chemically Synthesized Magnetite for Sustained Drug Delivery

Drug delivery using nanoparticles is attracting the attention of most researchers as it has got its targeted delivery, reduced side effects, and controlled release of drugs for a prolonged period of time is possible. In this study, magnetite was synthesized by a chemical method using the mixture of solutions of FeSO4 and FeCl3 as the iron source. The synthesized magnetite was subjected to analysis by FeSEM, XRD and VSM and hence characterized to have a size of 40-60nm and with a paraferromagnetic activity. The magnetite nanoparticle with the antibiotic rifampicin was coated with four different biopolymers, namely chitosan, starch, casein, and PHB. PHB coated nanoparticles were 80-90 nm-sized, which is the smallest size of all. The biopolymer coated nanoparticles were subjected to antimicrobial assay against E.coli by well diffusion method of which the PHA coated particles was found to be the best for holding rifampicin. The different biopolymer coated nanoparticles were also subjected to biofilm inhibition assay and MTT based cytotoxicity assay. Magnetite was found to have a LC50 at 60 µg/ml.

Antibacterial and antibiofilm effects of flufenamic acid against methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) infections are one of the most serious surgery complications, and their prevention is of utmost importance. Flufenamic acid is a non-steroid anti-inflammatory drug approved for clinical use to relieve inflammation and pain in rheumatoid arthritis patients. In this study, we explored the antibacterial efficacy of flufenamic acid and the mechanisms underlying this effect. By using minimal inhibitory concentration (MIC), time-kill, resistance induction assays, and the antibiotic synergy test, we demonstrated that flufenamic acid inhibited the growth of methicillin-resistant staphylococci and did not induce resistance when it was used at the MIC. Furthermore, flufenamic acid acted synergistically with the beta-lactam antibiotic oxacillin and did not show significant toxicity toward mammalian cells. The biofilm inhibition assay revealed that flufenamic acid could prevent biofilm formation on medical implants and destroy the ultrastructure of the bacterial cell wall. RNA sequencing and quantitative RT-PCR indicated that flufenamic acid inhibited the expression of genes associated with peptidoglycan biosynthesis, beta-lactam resistance, quorum sensing, and biofilm formation. Furthermore, flufenamic acid efficiently ameliorated a local infection caused by MRSA in mice. In conclusion, flufenamic acid may be a potent therapeutic compound against MRSA infections and a promising candidate for antimicrobial coating of implants and surgical devices.

Antibacterial and Anti-Biofilm Activities of Culture Filtrates from Schizophyllum commune, Coniothyrium sp., and Fusarium sp.

Bacterial infections are usually caused by biofilms that are resistant to extreme conditions. Studies have shown that fungal extracts have the potential to inhibit biofilm formation. The aim of this study was to examine the activity of several fungal culture filtrates in inhibiting bacterial growth and biofilm formation. The fungi were identified by molecular method, and the effects of different concentrations (45%, 75%, and 90%), medium pH (6, 7, and 9), temperatures (30 and 37 °C), and incubation times (24, 48, and 72 h) of the filtrates on their anti-biofilm formation properties were investigated. Anti-biofilm assay was performed using the diffusion test and microtiter assay. The parameters examined included the diameter of the inhibition zone of each well and the optical density of the filtrate solution. Molecular identification based on ITS rDNA regions showed that the fungal isolates in this study were Schizophyllum commune (strain JSB2), Coniothyrium sp. (strain JB1-3), and Fusarium sp. (strain JBB2). Antibacterial assay showed that, among the culture filtrates obtained from all fungi, that from Fusarium sp. strain JBB2 exhibits the highest inhibitory activity. Biofilm inhibition assay further revealed optimum fungal culture filtrate activities at pH 7.

Antimicrobial Susceptibility Testing of Metronidazole and Clindamycin against Gardnerella vaginalis in Planktonic and Biofilm Formation.

Background Bacterial vaginosis (BV), one of the most common vaginal ecosystem-related microbiologic syndromes, is the most common disorder in women of reproductive age. Gardnerella (G.) vaginalis is the predominant species causing this infection. Our aim was to compare the antimicrobial susceptibilities of metronidazole and clindamycin against G. vaginalis at planktonic and biofilm levels. Methods From September 2019 to October 2019, we recruited a total of 10 patients with BV who underwent gynecological examinations at Beijing Obstetrics and Gynecology Hospital. G. vaginalis isolates were obtained from the vagina and identified using their characteristic colony morphology. Sequence data of clinical G. vaginalis isolates were confirmed by comparing 16S rDNA sequences. Subsequently, clinical isolates were evaluated for antimicrobial susceptibilities in vitro to metronidazole and clindamycin at planktonic and biofilm levels. The minimum inhibitory concentration (MIC) for metronidazole and clindamycin was evaluated by antimicrobial susceptibility testing. The minimum biofilm eradication concentration (MBEC) was evaluated by the biofilm inhibition assay. Results Planktonic clinical isolates showed a significantly higher susceptibility rate (76.67%) and lower resistance rate (23.33%) to clindamycin than to metronidazole (susceptibility rate: 38.24%; resistance rate: 58.82%; P < 0.05 for both). Furthermore, in comparison to planktonic isolates, the minimum inhibitory concentration (MIC) of metronidazole was significantly higher for biofilm-forming isolates (7.3 ± 2.6 μg/mL vs. 72.4 ± 18.3 μg/mL; P=0.005); the resistance rate was 27.3%, and the minimum biofilm eradication concentration (MBEC) was >128 μg/mL. Moreover, the MIC of clindamycin was higher too for biofilm-forming isolates (0.099 ± 0.041 μg/mL vs. 23.7 ± 9.49 μg/mL; P=0.034); the resistance rate was 27.3%, and the MBEC of clindamycin was 28.4 ± 6.50 μg/mL. Conclusion Our results indicate that in comparison to metronidazole, clindamycin seems to be a better choice to tackle G. vaginalis as it exhibits a relatively higher susceptibility rate and lower resistance rate.

2-Hydroxy-4-methoxybenzaldehyde from Hemidesmus indicus is antagonistic to Staphylococcus epidermidis biofilm formation

Staphylococcus epidermidis (SE) is an opportunistic nosocomial pathogen that accounts for recalcitrant device-related infections worldwide. Owing to the growing interest in plants and their secondary metabolites targeting bacterial adhesion, this study was intended to uncover the anti-biofilm potential of Hemidesmus indicus and its major constituent 2-hydroxy-4-methoxybenzaldehyde (HMB) against SE. The minimum biofilm inhibitory concentration (MBIC) of H. indicus root extract and HMB were found to be 500 and 250 µg ml−1, respectively. The results of time-dependent biofilm inhibition and mature biofilm disruption assays confirmed that HMB targets initial cell adhesion. Furthermore, interference by HMB in the expression of adhesin genes (icaA, aap and bhp) and biofilm components was associated with an increased susceptibility of SE to oxidative stress and antibiotics. To conclude, this study reports for the first time HMB as a potential drug against SE biofilms.

Bactericidal Capacity of a Heterogeneous TiO2/ZnO Nanocomposite against Multidrug-Resistant and Non-Multidrug-Resistant Bacterial Strains Associated with Nosocomial Infections.

The surge of medical devices associated with nosocomial infection (NI) cases, especially by multidrug-resistant (MDR) bacterial strains, is one of the pressing issues of present health care systems. Metal oxide nanoparticles (MNPs) have become promising antibacterial agents against a wide range of bacterial strains. This work study is on the bactericidal capacity of heterogeneous TiO2/ZnO nanocomposites with different weight percentages and concentrations against common MDR and non-MDR bacterial strains. The profiles on disk diffusion, minimum inhibitory concentration, minimum bactericidal concentration, tolerance determination, time-kill, and biofilm inhibition assay were determined after 24 h of direct contact with the nanocomposite samples. Findings from this work revealed that the heterogeneous TiO2/ZnO nanocomposite with a 25T75Z weight ratio showed an optimal tolerance ratio against Gram-positive and -negative bacteria, indicating their bactericidal capacity. Further observation suggests that higher molar ratio of Zn2+ may possibly involve generation of active ion species that enhance bactericidal effect against Gram-positive bacterial strains, especially for the MDR strains. Nano-based technology using MNPs may provide a promising solution for the prevention and control of NIs. Further work on biocompatibility and cytotoxicity profiles of this nanocomposite are needed.

A Rational Designed PslG With Normal Biofilm Hydrolysis and Enhanced Resistance to Trypsin-Like Protease Digestion.

A glycosyl hydrolase produced by Pseudomonas aeruginosa, PslG, has become a promising candidate for biofilm treatment because of its ability to inhibit and disperse biofilms by disrupting exopolysaccharide matrix at nanomolar concentrations. However, as a protein, PslG used for treatment may be degraded by the ubiquitous proteases (of which trypsin-like serine proteases are a major group) secreted by human cells. This would lead to an insufficient effective concentration of PslG. Here, based on the result of liquid chromatography–tandem mass spectrometry (LC-MS/MS) and structural analysis, we generate a PslG mutant (K286A/K433S) with greatly enhanced trypsin resistance. This measure raises IC50 (the concentration of trypsin that can degrade 50% of protein in 30 min at 37°C) from 0.028 mg mL–1 of the wild-type PslG to 0.283 mg mL–1 of PslGK286A/K433S. In addition, biofilm inhibition assay shows that PslGK286A/K433S is much more efficient than wild-type PslG in the presence of trypsin. This indicates that PslGK286A/K433S is a better biofilm inhibitor than wild-type PslG in clinical use where trypsin-like proteases widely exist.

Characterization of biofilm formation and induction of apoptotic DNA fragmentation by nontypeable Haemophilus influenzae on polarized human airway epithelial cells

Nontypeable Haemophilus influenzae (NTHi) is a common airway commensal and opportunistic pathogen that persists within biofilm communities in vivo. Biofilm studies so far are mainly based on assays on plastic surfaces. The aim of this work was to investigate the capacity of clinical NTHi strains to form biofilm structures on polarized Calu-3 human airway epithelial cells and primary normal human bronchial epithelial cells and to characterize the biofilm architecture. Formation of adherent NTHi biofilms post colonization of host cells at multiple time-points was evaluated using confocal laser scanning microscopy and electron microscopy. NTHi biofilms were analyzed in terms of biofilm height and presence of extracellular matrix components, and their apoptotic effects on epithelial cells were measured by TUNEL assay. Strain Fi176 was observed to form robust biofilms on airway epithelia over time, while disrupting the integrity of Calu-3 monolayer by 72 h of co-culture. NTHi biofilms were observed to induce apoptotic DNA fragmentation in host cells at 24 h post infection. Biofilm formation on cell monolayers by Fi176ΔpilA strain was markedly reduced compared to WT strain. Biofilm inhibition and disruption assays by crystal violet staining indicated that DNA and proteins are part of NTHi biofilms in vitro. Our findings highlight critical stages of NTHi pathogenesis following host colonization and provide useful biofilm models for future antimicrobial drug discovery investigations.