Proteinase K/Retinoic Acid-Loaded Cationic Liposomes as Multifunctional Anti-Acne Therapy to Disorganize Biofilm and Regulate Keratinocyte Proliferation.

Background: Simultaneous anti-Cutibacterium acnes and anti-inflammatory actions are highly beneficial in treating acne vulgaris. In this study, we present novel anti-acne nanovesicles based on liposomes loaded with proteinase K (PK), retinoic acid (RA), and soyaethyl morpholinium ethosulfate (SME) to achieve an effective and safe treatment. Materials and Methods: This study examined in vitro planktonic and biofilm C. acnes elimination, as well as the keratinocyte proliferation suppression by liposomes. The multifunctional liposomes for treating C. acnes in mice were also evaluated. Results: We acquired multifunctional liposomes with a size of 71 nm and zeta potential of 31 mV. The antimicrobial activity of SME was enhanced after liposomal encapsulation according to the reduction of minimum bactericidal concentration (MBC) by 6-fold. The multifunctional liposomes exhibited a synergistically inhibitory effect on biofilm C. acnes colonization compared with the liposomes containing PK or those containing SME individually. The adhesive bacterial colony in the microplate was lessened by 62% after multifunctional liposome intervention. All liposomal formulations tested here demonstrated no cytotoxicity against the normal keratinocytes but inhibited C. acnes-stimulated cell hyperproliferation. The in vitro scratch assay indicated that the liposomal RA-but not free RA-restrained keratinocyte migration. The animal study showed that free RA combined with SME and multifunctional nanovesicles had a similar effect on diminishing C. acnes colonies in the skin. On the other hand, liposomes exhibited superior performance in recovering the impaired skin barrier function than the free control. We also found that RA-loaded nanovesicles had greater skin tolerability than free RA. Conclusion: The cationic liposomes containing dual PK and RA represented a potential treatment to arrest bacterial infection and associated inflammation in acne.

Evaluation of deoxythymidine-based cationic amphiphiles as antimicrobial, antibiofilm, and anti-inflammatory agents.

OBJECTIVES: We recently designed a series of cationic deoxythymidine-based amphiphiles that mimic the cationic amphipathic structure of antimicrobial peptides (AMPs). Among these amphiphiles, ADG-2e and ADL-3e displayed the highest selectivity against bacterial cells. In this study, ADG-2e and ADL-3e were evaluated for their potential as novel classes of antimicrobial, antibiofilm, and anti-inflammatory agents. METHODS: Minimum inhibitory concentrations of ADG-2e and ADL-3e against bacteria were determined using the broth microdilution method. Proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K was determined by radial diffusion and HPLC analysis. Biofilm activity was investigated using the broth microdilution and confocal microscopy. The antimicrobial mechanism was investigated by membrane depolarization, cell membrane integrity analysis, scanning electron microscopy (SEM), genomic DNA influence and genomic DNA binding assay. Synergistic activity was evaluated using checkerboard method. Anti-inflammatory activity was investigated using ELISA and RT-PCR. RESULTS: ADG-2e and ADL-3e showed good resistance to physiological salts and human serum, and a low incidence of drug resistance. Moreover, they exhibit proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K. ADG-2e and ADL-3e were found to kill bacteria by an intracellular target mechanism and bacterial cell membrane-disrupting mechanism, respectively. Furthermore, ADG-2e and ADL-3e showed effective synergistic effects when combined with several conventional antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Importantly, ADG-2e and ADL-3e not only suppressed MDRPA biofilm formation but also effectively eradicated mature MDRPA biofilms. Furthermore, ADG-2e and ADL-3e drastically decreased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) gene expression and protein secretion in lipopolysaccharide (LPS)-stimulated macrophages, implying potent anti-inflammatory activity in LPS-induced inflammation. CONCLUSION: Our findings suggest that ADG-2e and ADL-3e could be further developed as novel antimicrobial, antibiofilm, and anti-inflammatory agents to combat bacterial infections.

Antibioflm effects of extracellular matrix degradative agents on the biofilm of different strains of multi-drug resistant Corynebacterium striatum.

BACKGROUND: Corynebacterium striatum is a microorganism with an excellent capacity for biofilm production and thus has been correlated with nosocomial transmission and invasive infections. However, little is known about the mechanism of biofilm formation of this commensal pathogen. In this study, we aimed to investigate the biofilm formation abilities of multidrug-resistant Corynebacterium striatum clinical isolates and the roles of extracellular proteins, exopolysaccharides and extracellular DNA in mediating more robust biofilm formation by the isolates of C. striatum. METHODS: C. striatum isolates were identified using VITEK-2 ANC card, matrix-assisted laser desorption/ionization-time of flight mass spectrometry and 16S rRNA sequencing. The antibiotic susceptibility test was performed using the broth microdilution method. The distribution of spaDEF genes among C. striatum isolates was detected by polymerase chain reaction, and pulsed-field gel electrophoresis typing was employed to analyze the genotypes of the isolates. Crystal violet staining and scanning electron microscopy techniques were used to detect biofilm production by C. striatum isolates. Biofilm degradation assay was performed to observe the effects of extracellular matrix degradative agents (proteinase K, dispersin B, and DNase I) on C. striatum biofilms. RESULTS: Twenty-seven C. striatum isolates were enrolled in the study, and the resistance rates were the highest (100%, 27/27) against penicillin and ceftriaxone. Approximately 96.3% (26/27) C. striatum isolates were resistant to at least three different types of antimicrobial agents tested. All isolates were confirmed to be biofilm producers, and 74.07% (20/27) isolates presented moderate to strong biofilm production abilities. P7 genotype (44.4%, 12/27) was identified to as the predominant genotype, and all of isolates belonging to this genotype were multidrug-resistant and had stronger biofilm-forming abilities. Most C. striatum isolates (74.07%, 20/27) carry spaD, spaE, and spaF genes, which encode spa-type pili. However, the correlation between the expression of spa-type genes and the biofilm production abilities of the C. striatum isolates was not found. The biofilms of 80% (8/10), 90% (9/10), and 100% (10/10) C. striatum isolates with moderate to strong biofilm production abilities were significantly eliminated upon the treatment of dispersin B (20 µg/mL), DNase I (20 µg/mL), and proteinase K (20 µg/mL) (p < 0.05), respectively. For the combination groups with two kinds of biofilm-degradative agents, the combination of 20 µg/mL proteinase K/dispersin B showed the strongest biofilm-eliminating effects, when the biofilms of 90% (9/10) C. striatum isolates degraded more than 50%. CONCLUSIONS: The C. striatum isolates that belonged to the predominant genotype showed a multidrug resistance (MDR) phenotype and strong biofilm formation abilities. Extracellular matrix seems to be an essential determinant in mediating biofilm formation of MDR C. striatum, since extracellular matrix degradative agents (proteinase K, dispersin B and DNase I) showed strong biofilm-eliminating effects toward multidrug-resistant C. striatum isolates. The findings of this study highlight new ideas/directions to explore the whole nature of biofilm formation of C. striatum and the function of extracellular matrix in this process.

Decorating hexahistidine-metal assemblies with tyrosine enhances the ability of proteins to pass through corneal biobarriers.

In recent decades, the use of protein drugs has increased dramatically for almost every clinical indication, including autoimmunity and cancer infection, given their high specificity and limited side effects. However, their easy deactivation by the surrounding microenvironment and limited ability to pass through biological barriers pose large challenges to the use of these agents for therapeutic effects; these deficits could be greatly improved by nanodelivery using platforms with suitable physicochemical properties. Here, to assess the effect of the hydrophobicity of nanoparticles on their ability to penetrate biological barriers, the hydrophobic amino acid tyrosine (Y) was decorated onto hexahistidine peptide, and two nanosized YHmA and HmA particles were generated, in which Avastin (Ava, a protein drug) was encapsulated by a coassembly strategy. In vitro and in vivo tests demonstrated that these nanoparticles effectively retained the bioactivity of Ava and protected Ava from proteinase K hydrolysis. Importantly, YHmA displayed a considerably higher affinity to the ocular surface than HmA, and YHmA also exhibited the ability to transfer proteins across the barriers of the anterior segment, which greatly improved the bioavailability of the encapsulated Ava and produced surprisingly good therapeutic outcomes in a model of corneal neovascularization. STATEMENT OF SIGNIFICANCE: Improving the ability to penetrate tissue barriers and averting inactivation caused by surrounding environments, are the keys to broaden the application of protein drugs. By decorating hydrophobic amino acid, tyrosine (Y), on hexahistidine peptide, YHmA encapsulated protein drug Ava with high efficiency by co-assembly strategy. YHmA displayed promising ability to maintain bioactivity of Ava during encapsulation and delivery, and protected Ava from proteinase K hydrolysis. Importantly, YHmA transferred Ava across the corneal epithelial barrier and greatly improved its bioavailability, producing surprisingly good therapeutic outcomes in a model of corneal neovascularization. Our results contributed to not only the strategy to overcome shortcomings of protein drugs, but also suggestion on hydrophobicity as a nonnegligible factor in nanodrug penetration through biobarriers.

Novel D-form of hybrid peptide (D-AP19) rapidly kills Acinetobacter baumannii while tolerating proteolytic enzymes.

Antimicrobial peptides (AMPs) are being developed as potent alternative treatments to conventional antibiotics which are unlikely to induce bacterial resistance. They can be designed and modified to possess several druggable properties. We report herein a novel hybrid peptide of modified aurein (A3) and cathelicidin (P7), or A3P7, by a flipping technique. It exhibited potent antibacterial activity against both Gram-negative and -positive pathogenic bacteria but had moderate hemolytic activity. To reduce the sequence length and toxicity, C-terminal truncation was serially performed and eight truncated derivatives (AP12-AP19) were obtained. They had significantly less hemolytic activity while preserving antibacterial activity. Secondary structures of the candidate peptides in environments simulating bacterial membranes (30 mM SDS and 50% TFE), determined by CD spectroscopy, showed α-helical structures consistent with predicted in silico 3D structural models. Among the peptides, AP19 demonstrated the best combination of broad-spectrum antibacterial activity (including toward Acinetobacter baumannii) and minimal hemolytic and cytotoxic activities. A D-form peptide (D-AP19), in which all L-enantiomers were substituted with the D-enantiomers, maintained antibacterial activity in the presence of pepsin, trypsin, proteinase K and human plasma. Both isomers exhibited potent antibacterial activity against multi-drug (MDR) and extensively-drug resistant (XDR) clinical isolates of A. baumannii comparable to the traditional antibiotic, meropenem. D-AP19 displayed rapid killing via membrane disruption and leakage of intracellular contents. Additionally, it showed a low tendency to induce bacterial resistance. Our work suggested that D-AP19 could be further optimized and developed as a novel compound potentially for fighting against MDR or XDR A. baumannii.

Characterization of Staphylococcus aureus biofilms via crystal violet binding and biochemical composition assays of isolates from hospitals, raw meat, and biofilm-associated gene mutants.

Staphylococcus aureus (SA) is a gram-positive coccus and an opportunistic pathogen of humans. The ability of SA to form biofilms is an important virulence mechanism because biofilms are protected from host immune responses and antibiotic treatment. This study examines the relative biofilm strength of a variety of hospital and meat-associated strains of SA, using a crystal violet (CV) staining assay. Biofilms were treated with either DNase or proteinase K prior to CV staining, and compared to mock-treated results, to better understand the biochemical composition. Biofilm polysaccharide concentration was also measured using the phenol sulfuric-acid assay which was normalized to base biofilm strength. We found that hospital-associated isolates have biofilms that bind significantly more CV than for meat isolates and are significantly more protein and polysaccharide-based while meat isolates have significantly more DNA-based biofilms. This study also investigates the effects that biofilm-related genes have on biofilm formation and composition by analyzing specific transposon mutants of genes previously shown to play a role in biofilm development. agrA, atl, clfA, fnbA, purH, and sarA mutants produce significantly weaker biofilms (bind less CV) as compared to a wild-type control, whereas the acnA mutant produces a significantly stronger biofilm. Biofilms formed from these mutant strains were treated (or mock-treated) with DNase or proteinase K and tested with phenol and sulfuric acid to determine what role these genes play in biofilm composition. The acnA, clfA, fnbA, and purH mutants showed significant reduction in biofilm staining after either proteinase K or DNase treatment, agrA and sarA mutants showed significant biofilm reduction after only proteinase K treatment, and an atl mutant did not show significant biofilm reduction after either proteinase K or DNase treatment. These data suggest that biofilms that form without acnA, clfA, fnbA, and purH are DNA- and protein-based, that biofilms lacking agrA and sarA are mainly protein-based, and biofilms lacking atl are mainly polysaccharide-based. These results help to elucidate how these genes affect biofilm formation and demonstrate how mutating biofilm-related genes in SA can cause a change in biofilm composition.

Disease Inhibiting Effect of Strain Bacillus subtilis EG21 and Its Metabolites Against Potato Pathogens Phytophthora infestans and Rhizoctonia solani.

Potato production worldwide is plagued by several disease-causing pathogens that result in crop and economic losses estimated to billions of dollars each year. To this day, synthetic chemical applications remain the most widespread control strategy despite their negative effects on human and environmental health. Therefore, obtainment of superior biocontrol agents or their naturally produced metabolites to replace fungicides or to be integrated into practical pest management strategies has become one of the main targets in modern agriculture. Our main focus in the present study was to elucidate the antagonistic potential of a new strain identified as Bacillus subtilis EG21 against potato pathogens Phytophthora infestans and Rhizoctonia solani using several in vitro screening assays. Microscopic examination of the interaction between EG21 and R. solani showed extended damage in fungal mycelium, while EG21 metabolites displayed strong anti-oomycete and zoosporecidal effect on P. infestans. Mass spectrometry (MS) analysis revealed that EG21 produced antifungal and anti-oomycete cyclic lipopeptides surfactins (C12 to C19). Further characterization of EG21 confirmed its ability to produce siderophores and the extracellular lytic enzymes cellulase, pectinase and chitinase. The antifungal activity of EG21 cell-free culture filtrate (CF) was found to be stable at high-temperature/pressure treatment and extreme pH values and was not affected by proteinase K treatment. Disease-inhibiting effect of EG21 CF against P. infestans and R. solani infection was confirmed using potato leaves and tubers, respectively. Biotechnological applications of using microbial agents and their bioproducts for crop protection hold great promise to develop into effective, environment-friendly and sustainable biocontrol strategies. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Inhibitory effect of proteinase K against dermatophyte biofilms: an alternative for increasing the antifungal effects of terbinafine and griseofulvin.

This study aimed to evaluate the effect of proteinase K on mature biofilms of dermatophytes, by assays of metabolic activity and biomass. In addition, the proteinase K-terbinafine and proteinase K-griseofulvin interactions against these biofilms were investigated by the checkerboard assay and scanning electron and confocal microscopy. The biofilms exposed to 32 µg ml-1 of proteinase K had lower metabolic activity and biomass, by 39% and 38%, respectively. Drug interactions were synergistic, with proteinase K reducing the minimum inhibitory concentration of antifungals against dermatophyte biofilms at a concentration of 32 µg ml-1 combined with 128-256 µg ml-1 of terbinafine and griseofulvin. Microscopic images showed a reduction in biofilms exposed to proteinase K, proteinase K-terbinafine and proteinase K-griseofulvin combinations. These findings demonstrate that proteinase K has activity against biofilms of dermatophytes, and the interactions of proteinase K with terbinafine and griseofulvin improve the activity of drugs against mature dermatophyte biofilms.

Serum circulating proteins from pediatric patients with dilated cardiomyopathy cause pathologic remodeling and cardiomyocyte stiffness.

Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy and main indication for heart transplantation in children. Therapies specific to pediatric DCM remain limited due to lack of a disease model. Our previous study showed that treatment of neonatal rat ventricular myocytes (NRVMs) with serum from nonfailing or DCM pediatric patients activates the fetal gene program (FGP). Here we show that serum treatment with proteinase K prevents activation of the FGP, whereas RNase treatment exacerbates it, suggesting that circulating proteins, but not circulating miRNAs, promote these pathological changes. Evaluation of the protein secretome showed that midkine (MDK) is upregulated in DCM serum, and NRVM treatment with MDK activates the FGP. Changes in gene expression in serum-treated NRVMs, evaluated by next-generation RNA-Seq, indicated extracellular matrix remodeling and focal adhesion pathways were upregulated in pediatric DCM serum and in DCM serum-treated NRVMs, suggesting alterations in cellular stiffness. Cellular stiffness was evaluated by Atomic Force Microscopy, which showed an increase in stiffness in DCM serum-treated NRVMs. Of the proteins increased in DCM sera, secreted frizzled-related protein 1 (sFRP1) was a potential candidate for the increase in cellular stiffness, and sFRP1 treatment of NRVMs recapitulated the increase in cellular stiffness observed in response to DCM serum treatment. Our results show that serum circulating proteins promoted pathological changes in gene expression and cellular stiffness, and circulating miRNAs were protective against pathological changes.

Co-Cultivation of Two Bacillus Strains for Improved Cell Growth and Enzyme Production to Enhance the Degradation of Aflatoxin B1.

Bacillus sp. H16v8 and Bacillus sp. HGD9229 were identified as Aflatoxin B1 (AFB1) degrader in nutrient broth after a 12 h incubation at 37 °C. The degradation efficiency of the two-strain supernatant on 100 µg/L AFB1 was higher than the bacterial cells and cell lysate. Moreover, degradations of AFB1 were strongly affected by the metal ions in which Cu2+ stimulated the degradation and Zn2+ inhibited the degradation. The extracellular detoxifying enzymes produced by co-cultivation of two strains were isolated and purified by ultrafiltration. The molecular weight range of the detoxifying enzymes was 20-25 kDa by SDS-PAGE. The co-culture of two strains improved the total cell growth with the enhancement of the total protein content and detoxifying enzyme production. The degradation efficiency of the supernatant from mixed cultures increased by 87.7% and 55.3% compared to Bacillus sp. H16v8 and HGD9229, individually. Moreover, after the degradation of AFB1, the four products of the lower toxicity were identified by LC-Triple TOF-MS with the two proposed hypothetical degradation pathways.

Strain variation in Bacillus cereus biofilms and their susceptibility to extracellular matrix-degrading enzymes.

Bacillus cereus is a foodborne pathogen and can form biofilms on food contact surfaces, which causes food hygiene problems. While it is necessary to understand strain-dependent variation to effectively control these biofilms, strain-to-strain variation in the structure of B. cereus biofilms is poorly understood. In this study, B. cereus strains from tatsoi (BC4, BC10, and BC72) and the ATCC 10987 reference strain were incubated at 30°C to form biofilms in the presence of the extracellular matrix-degrading enzymes DNase I, proteinase K, dispase II, cellulase, amyloglucosidase, and α-amylase to assess the susceptibility to these enzymes. The four strains exhibited four different patterns in terms of biofilm susceptibility to the enzymes as well as morphology of surface-attached biofilms or suspended cell aggregates. DNase I inhibited the biofilm formation of strains ATCC 10987 and BC4 but not of strains BC10 and BC72. This result suggests that some strains may not have extracellular DNA, or their extracellular DNA may be protected in their biofilms. In addition, the strains exhibited different patterns of susceptibility to protein- and carbohydrate-degrading enzymes. While other strains were resistant, strains ATCC 10987 and BC4 were susceptible to cellulase, suggesting that cellulose or its similar polysaccharides may exist and play an essential role in their biofilm formation. Our compositional and imaging analyses of strains ATCC 10987 and BC4 suggested that the physicochemical properties of their biofilms are distinct, as calculated by the carbohydrate to protein ratio. Taken together, our study suggests that the extracellular matrix of B. cereus biofilms may be highly diverse and provides insight into the diverse mechanisms of biofilm formation among B. cereus strains.

Proteinase K treatment in absence of RNA isolation classical procedures is a quick and cheaper alternative for SARS-CoV-2 molecular detection.

The World Health Organization (WHO) has declared a pandemic of COVID-19, the disease caused by the recently described SARS-CoV-2. The relevance and importance of mass diagnosis in order to find the asymptomatic individuals is widely recognized as a mandatory tool to reinforce the control measures for monitoring virus circulation and reduce the spreading of SARS-CoV-2. Here, we described quickness and cheaper strategies of direct RT-qPCR (in the absence of RNA isolation) and compared the results to those obtained using standard RNA isolation procedure. The tests varied using pure, diluted samples, combined with Proteinase K (PK) or Lysis Buffer. Our findings showed consistently that PK pre-treated samples in the absence of RNA extraction procedures presents similar results to those obtained by standard RNA isolation procedures. On average, 16 samples extracted with the MagMAX™ CORE Kit, take around 2 h, costing an average of USD 5, the pre-treatment of samples using PK, on the other hand, would cut the value to less than USD 0.30 and reduce the time of procedure in more than 1 ½ hours. The present study suggests the use of PK treatment instead of RNA isolation in order to reduce costs and time in processing samples for molecular diagnosis of SARS-CoV-2.

Synergistic anti-biofilm effects of Brassicaceae plant extracts in combination with proteinase K against Escherichia coli O157:H7.

Bacteria can form biofilms, complex microbial communities protected from environmental stress, on food contact surfaces. Brassicaceae plant has been shown to contain bioactive compounds with antimicrobial activities. The objective of this study was to evaluate the synergistic effects of Brassicaceae species and proteinase K against E. coli O157:H7 biofilm. We determined the minimum biofilm inhibitory concentration, the fractional inhibitory concentration indexes, and the synergistic inhibitory effect of Raphanus sativus var. longipinnatus, R. sativus, and Brassica oleracea var. acephala extracts with proteinase K on E. coli O157:H7. The biofilm showed a 49% reduction with 2 mg/mL R. sativus. The combination of proteinase K 25 µg/mL significantly increased the effect of 2 mg/mL R. sativus var. longipinnatus and the combined treatment yielded up to 2.68 log reduction on stainless steel coupons. The results showed that the combination of R. sativus var. longipinnatus extract and proteinase K could serve as an anti-biofilm agent with synergistic effects for inhibiting E. coli O157:H7 biofilm on stainless steel surfaces.

Enzymatic and Chemical-Based Methods to Inactivate Endogenous Blood Ribonucleases for Nucleic Acid Diagnostics.

There are ongoing research efforts into simple and low-cost point-of-care nucleic acid amplification tests (NATs) addressing widespread diagnostic needs in resource-limited clinical settings. Nucleic acid testing for RNA targets in blood specimens typically requires sample preparation that inactivates robust blood ribonucleases (RNases) that can rapidly degrade exogenous RNA. Most NATs rely on decades-old methods that lyse pathogens and inactivate RNases with high concentrations of guanidinium salts. Herein, we investigate alternatives to standard guanidinium-based methods for RNase inactivation using an activity assay with an RNA substrate that fluoresces when cleaved. The effects of proteinase K, nonionic surfactants, SDS, dithiothreitol, and other additives on RNase activity in human serum are reported. Although proteinase K has been widely used in protocols for nuclease inactivation, it was found that high concentrations of proteinase K are unable to eliminate RNase activity in serum, unless used in concert with denaturing concentrations of SDS. It was observed that SDS must be combined with proteinase K, dithiothreitol, or both for irreversible and complete RNase inactivation in serum. This work provides an alternative chemistry for inactivating endogenous RNases for use in simple, low-cost point-of-care NATs for blood-borne pathogens.

Presence of extracellular DNA & protein in biofilm formation by gentamicin-resistant Lactobacillus plantarum.

Background & objectives: Bacterial biofilms a multi-layered defence, comprise extracellular DNA (eDNA) and proteins, protect bacteria from harmful environment and nutrient limitation and utilize the mutual benefits within a community. Bacterial biofilms also defend bacteria from harsh environments such as antibiotic treatment. This leads to poor antibiotic penetration, slow growth, adaptive stress responses, and formation of persister cells. This study was done to determine the relation of antibiotic resistance deciphered by the biofilms in Lactobacillus plantarum, a lactic acid bacteria (LAB) with probiotic significance. Methods: The gentamicin-resistant L. plantarum isolates were allowed to form biofilms and subjected to DNase I and proteinase K treatment. The optical density (OD) values were recorded for the biofilm assay and the cell count for the number of viable cells was taken for the control and the test samples. Percentage reduction was calculated based on the difference between the initial and final OD for both the parameters. Results: The biofilm assay revealed that the native L. plantarum isolates which were phenotypically susceptible, possessed the ability to form biofilms. The OD values were significantly decreased in comparison to the biofilm-forming control culture when these were treated with DNase I and proteinase K. Interpretation & conclusions: The study revealed that the biofilms formed by L. plantarum comprised of eDNA and proteins which was evidenced by the reduction in OD values and percentage in comparison to the control upon DNase I and proteinase K treatment. This indicates that the eDNA and biofilm matrix proteins are vital constituents of biofilms and may carry significant risk when coupled with antibiotic resistance.

An Aggregation-defective Mutant of Methanothermobacter sp. CaT2 Reveals Unique Protein-dependent Aggregation.

The thermophilic hydrogenotrophic methanogen, Methanothermobacter sp. CaT2, which possesses an extracellular sugar layer, commonly aggregates by itself or with other microorganisms. To elucidate the molecular mechanisms responsible for this aggregation, the aggregation-defective mutant, CLA160, was isolated. Optical and electron microscopy observations revealed that the mutant exhibited a significant reduction in aggregation. Genomic sequencing showed that CLA160 has a single point mutation, causing a nonsense mutation in MTCT_1020, which encodes a hypothetical protein. Motif and domain analyses indicated that the hypothetical protein bears two membrane-spanning segments at the N- and C-terminal regions and a large middle repeat-containing region. The results of a bioinformatic analysis suggested that the first middle region (RII) of the protein or the whole structure is responsible for the function of the product of MTCT_1020 in the aggregation of CaT2. A treatment with proteinase K suppressed sedimentation in CaT2, indicating a reduction in aggregation, with almost no effect on sedimentation in CLA160. The addition of Ca2+ or Mg2+ ions enhanced sedimentation in CaT2, whereas a DNase treatment had no effect on sedimentation in either strain. These results suggest that the hypothetical protein encoded by MTCT_1020 plays a key role as a membrane-bound adhesion protein in the aggregation of CaT2, which is enhanced by the addition of Ca2+ or Mg2+ ions.

Anti-genotoxic and anti-mutagenic activity of Escherichia coli Nissle 1917 as assessed by in vitro tests.

Anti-genotoxic or anti-mutagenic activity has been described for a number of Gram-positive probiotic bacterial species. Here we present evidence that Gram-negative Escherichia coli Nissle 1917 (EcN) also displays anti-genotoxic/anti-mutagenic activity, as assessed in vitro by the Comet Assay and the Ames Test, respectively. This activity was demonstrated by use of the mutagens 4-nitroquinoline-1-oxide (NQO), hydrogen peroxide (H2O2) and benzo(a) pyrene (B[a]P). For both assays and all three test agents the anti-genotoxic/anti-mutagenic activity of EcN was shown to be concentration dependent. By the use of extracts of bacteria that were inactivated by various procedures (heat treatment, ultrasound sonication or ultraviolet light irradiation), mechanistic explanations could be put forward. The proposed mechanisms were enforced by treating the bacterial material with proteinase K prior to testing. The mutagen H2O2 is most likely inactivated by enzymic activity, with catalase a likely candidate, while several explanations can be put forward for inactivation of B[a]P. NQO is most likely inactivated by metabolising enzymes, since the formation of the metabolite 4-aminoquinoline could be demonstrated. In conclusion, the in vitro results presented here make a strong case for antimutagenic properties of EcN.

Lactic Acid Produced by Glycolysis Contributed to Staphylococcus aureus Aggregation Induced by Glucose.

High concentration of glucose induces Staphylococcus aureus (S. aureus) aggregation, but the mechanism of this is still unclear. In this study, the aggregation of S. aureus strains was induced by high concentration of glucose (>7.8 mM), and which was dose- and time-dependent. In addition, the large amount of lactate acid produced during S. aureus aggregation, induced by glucose, resulted in decreased pH value. Lactic acid, the end product of glycolysis, could quickly induce S. aureus aggregation. Except for lactic acid, acetic acid and HCl also induced S. aureus aggregation. In addition, the aggregation of S. aureus strains induced by glucose or lactic acid was completely inhibited in Tris-HCl buffer (pH 7.5), and inhibition of glycolysis by 2-deoxyglucose significantly decreased S. aureus aggregation. The aggregation induced by glucose was dispersed by periodate and proteinase K. In summary, lactate acid produced by glycolysis contributed to S. aureus aggregation induced by high concentration of glucose.

Microrheology and Spatial Heterogeneity of Staphylococcus aureus Biofilms Modulated by Hydrodynamic Shear and Biofilm-Degrading Enzymes.

Particle tracking microrheology was used to investigate the viscoelasticity of Staphylococcus aureus biofilms grown in microfluidic cells at various flow rates and when subjected to biofilm-degrading enzymes. Biofilm viscoelasticity was found to harden as a function of shear rate but soften with increasing height away from the attachment surface in good agreement with previous bulk results. Ripley's K-function was used to quantify the spatial distribution of the bacteria within the biofilm. For all conditions, biofilms would cluster as a function of height during growth. The effects of proteinase K and DNase-1 on the viscoelasticity of biofilms were also investigated. Proteinase K caused an order of magnitude change in the compliances, softening the biofilms. However, DNase-1 was found to have no significant effects over the first 6 h of development, indicating that DNA is less important in biofilm maintenance during the initial stages of growth. Our results demonstrate that during the preliminary stages of Staphylococcus aureus biofilm development, column-like structures with a vertical gradient of viscoelasticity are established and modulated by the hydrodynamic shear caused by fluid flow in the surrounding environment. An understanding of these mechanical properties will provide more accurate insights for removal strategies of early-stage biofilms.

Characterization of a novel bacteriophage specific to Exiguobacterium indicum isolated from a plateau eutrophic lake.

Exiguobacterium is a versatile genus with potential in industry and agriculture. No bacteriophage that infects Exiguobacterium has been reported, despite its potential impacts on the utilization of Exiguobacterium. E. indicum EI9 was isolated from Dianchi Lake, a plateau eutrophic lake in China, which can significantly inhibit the growth of Microcystis aeruginosa. To isolate and characterize Exiguobacterium-infecting bacteriophage, a virulent bacteriophage, DCEIV-9 that specifically infects E. indicum EI9 was isolated from Dianchi lake water sample. DCEIV-9 produced tiny, round, and clear plaques with 0.5-1 mm in diameter. Electron microscopy showed that DCEIV-9 is a typical representative of the Siphoviridae, with an icosahedral head (56 nm in diameter) and a non-contractile tail (163 nm in length). Based on a one-step growth curve, latent period of 20 min and burst size of 51 PFU/infected cell were determined. DCEIV-9 was sensitive to temperature over 50 °C and prefers acid environment. DCEIV-9 was extremely sensitive to proteinase K, chloroform, ethanol, Triton X-100 but not sensitive to SDS. Restriction endonucleases analysis indicated that DCEIV-9 is a dsDNA virus. DCEIV-9 can only infect E. indicum, indicates that it has a narrow host range. DCEIV-9 is a potential new species.