ResearchPad - biofilms https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Confocal Raman microscopy to identify bacteria in oral subgingival biofilm models]]> https://www.researchpad.co/article/elastic_article_7675 The study of oral disease progression, in relation to the accumulation of subgingival biofilm in gingivitis and periodontitis is limited, due to either the ability to monitor plaque in vitro. When compared, optical spectroscopic techniques offer advantages over traditional destructive or biofilm staining approaches, making it a suitable alternative for the analysis and continued development of three-dimensional structures. In this work, we have developed a confocal Raman spectroscopy analysis approach towards in vitro subgingival plaque models. The main objective of this study was to develop a method for differentiating multiple oral subgingival bacterial species in planktonic and biofilm conditions, using confocal Raman microscopy. Five common subgingival bacteria (Fusobacterium nucleatum, Streptococcus mutans, Veillonella dispar, Actinomyces naeslundii and Prevotella nigrescens) were used and differentiated using a 2-way orthogonal Partial Least Square with Discriminant Analysis (O2PLS-DA) for the collected spectral data. In addition to planktonic growth, mono-species biofilms cultured using the ‘Zürich Model’ were also analyzed. The developed method was successfully used to predict planktonic and mono-species biofilm species in a cross validation setup. The results show differences in the presence and absence of chemical bands within the Raman spectra. The O2PLS-DA model was able to successfully predict 100% of all tested planktonic samples and 90% of all mono-species biofilm samples. Using this approach we have shown that Confocal Raman microscopy can analyse and predict the identity of planktonic and mono-species biofilm species, thus enabling its potential as a technique to map oral multi-species biofilm models.

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<![CDATA[Abrogation of pathogenic attributes in drug resistant <i>Candida auris</i> strains by farnesol]]> https://www.researchpad.co/article/elastic_article_7651 Candida auris, a decade old Candida species, has been identified globally as a significant nosocomial multidrug resistant (MDR) pathogen responsible for causing invasive outbreaks. Biofilms and overexpression of efflux pumps such as Major Facilitator Superfamily and ATP Binding Cassette are known to cause multidrug resistance in Candida species, including C. auris. Therefore, targeting these factors may prove an effective approach to combat MDR in C. auris. In this study, 25 clinical isolates of C. auris from different hospitals of South Africa were used. All the isolates were found capable enough to form biofilms on 96-well flat bottom microtiter plate that was further confirmed by MTT reduction assay. In addition, these strains have active drug efflux mechanism which was supported by rhodamine-6-G extracellular efflux and intracellular accumulation assays. Antifungal susceptibility profile of all the isolates against commonly used drugs was determined following CLSI recommended guidelines. We further studied the role of farnesol, an endogenous quorum sensing molecule, in modulating development of biofilms and drug efflux in C. auris. The MIC for planktonic cells ranged from 62.5–125 mM, and for sessile cells was 125 mM (4h biofilm) and 500 mM (12h and 24h biofilm). Furthermore, farnesol (125 mM) also suppresses adherence and biofilm formation by C. auris. Farnesol inhibited biofilm formation, blocked efflux pumps and downregulated biofilm- and efflux pump- associated genes. Modulation of C. auris biofilm formation and efflux pump activity by farnesol represent a promising approach for controlling life threatening infections caused by this pathogen.

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<![CDATA[Chitosan-propolis nanoparticle formulation demonstrates anti-bacterial activity against Enterococcus faecalis biofilms]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdc122

Propolis obtained from bee hives is a natural substance with antimicrobial properties. It is limited by its insolubility in aqueous solutions; hence ethanol and ethyl acetate extracts of Malaysian propolis were prepared. Both the extracts displayed antimicrobial and anti-biofilm properties against Enterococcus faecalis, a common bacterium associated with hospital-acquired infections. High performance liquid chromatography (HPLC) analysis of propolis revealed the presence of flavonoids like kaempferol and pinocembrin. This study investigated the role of propolis developed into nanoparticles with chitosan for its antimicrobial and anti-biofilm properties against E. faecalis. Bacteria that grow in a slimy layer of biofilm are resistant to penetration by antibacterial agents. The use of nanoparticles in medicine has received attention recently due to better bioavailability, enhanced penetrative capacity and improved efficacy. A chitosan-propolis nanoformulation was chosen based on ideal physicochemical properties such as particle size, zeta potential, polydispersity index, encapsulation efficiency and the rate of release of the active ingredients. This formulation inhibited E. faecalis biofilm formation and reduced the number of bacteria in the biofilm by ~90% at 200 μg/ml concentration. When tested on pre-formed biofilms, the formulation reduced bacterial number in the biofilm by ~40% and ~75% at 200 and 300 μg/ml, respectively. The formulation not only reduced bacterial numbers, but also physically disrupted the biofilm structure as observed by scanning electron microscopy. Treatment of biofilms with chitosan-propolis nanoparticles altered the expression of biofilm-associated genes in E. faecalis. The results of this study revealed that chitosan-propolis nanoformulation can be deemed as a potential anti-biofilm agent in resisting infections involving biofilm formation like chronic wounds and surgical site infections.

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<![CDATA[Investigation of synovial fluid induced Staphylococcus aureus aggregate development and its impact on surface attachment and biofilm formation]]> https://www.researchpad.co/article/Nf13f73b5-5132-41b9-b894-3d4dd0a113b1

Periprosthetic joint infections (PJIs) are a devastating complication that occurs in 2% of patients following joint replacement. These infections are costly and difficult to treat, often requiring multiple corrective surgeries and prolonged antimicrobial treatments. The Gram-positive bacterium Staphylococcus aureus is one of the most common causes of PJIs, and it is often resistant to a number of commonly used antimicrobials. This tolerance can be partially attributed to the ability of S. aureus to form biofilms. Biofilms associated with the surface of indwelling medical devices have been observed on components removed during chronic infection, however, the development and localization of biofilms during PJIs remains unclear. Prior studies have demonstrated that synovial fluid, in the joint cavity, promotes the development of bacterial aggregates with many biofilm-like properties, including antibiotic resistance. We anticipate these aggregates have an important role in biofilm formation and antibiotic tolerance during PJIs. Therefore, we sought to determine specifically how synovial fluid promotes aggregate formation and the impact of this process on surface attachment. Using flow cytometry and microscopy, we quantified the aggregation of various clinical S. aureus strains following exposure to purified synovial fluid components. We determined that fibrinogen and fibronectin promoted bacterial aggregation, while cell free DNA, serum albumin, and hyaluronic acid had minimal effect. To determine how synovial fluid mediated aggregation affects surface attachment, we utilized microscopy to measure bacterial attachment. Surprisingly, we found that synovial fluid significantly impeded bacterial surface attachment to a variety of materials. We conclude from this study that fibrinogen and fibronectin in synovial fluid have a crucial role in promoting bacterial aggregation and inhibiting surface adhesion during PJI. Collectively, we propose that synovial fluid may have conflicting protective roles for the host by preventing adhesion to surfaces, but by promoting bacterial aggregation is also contributing to the development of antibiotic tolerance.

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<![CDATA[Potential combinations of endocannabinoid/endocannabinoid-like compounds and antibiotics against methicillin-resistant Staphylococcus aureus]]> https://www.researchpad.co/article/Ne8a72c2e-13c7-43d3-9f49-0ed6410d9d0b

Infections caused by antibiotic-resistant strains of Staphylococcus aureus have reached epidemic proportions globally. Our previous study showed antimicrobial effects of anandamide (AEA) and arachidonoyl serine (AraS) against methicillin (MET)-resistant S. aureus (MRSA) strains, proposing the therapeutic potential of these endocannabinoid/endocannabinoid-like (EC/EC-like) agents for the treatment of MRSA. Here, we investigated the potential synergism of combinations of AEA and AraS with different types of antibiotics against MRSA grown under planktonic growth or biofilm formation. The most effective combinations under planktonic conditions were mixtures of AEA and ampicillin (AMP), and of AraS and gentamicin (GEN). The combination with the highest synergy in the biofilm formation against all tested bacterial strains was AEA and MET. Moreover, the combination of AraS and MET synergistically caused default of biofilm formation. Slime production of MRSA was also dramatically impaired by AEA or AraS combined with MET. Our data suggest the novel potential activity of combinations of EC/EC-like agents and antibiotics in the prevention of MRSA biofilm formation.

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<![CDATA[Cationic chitosan-propolis nanoparticles alter the zeta potential of S. epidermidis, inhibit biofilm formation by modulating gene expression and exhibit synergism with antibiotics]]> https://www.researchpad.co/article/5c818e84d5eed0c484cc2423

Staphylococcus epidermidis, is a common microflora of human body that can cause opportunistic infections associated with indwelling devices. It is resistant to multiple antibiotics necessitating the need for naturally occurring antibacterial agents. Malaysian propolis, a natural product obtained from beehives exhibits antimicrobial and antibiofilm properties. Chitosan-propolis nanoparticles (CPNP) were prepared using Malaysian propolis and tested for their effect against S. epidermidis. The cationic nanoparticles depicted a zeta potential of +40 and increased the net electric charge (zeta potential) of S. epidermidis from -17 to -11 mV in a concentration-dependent manner whereas, ethanol (Eth) and ethyl acetate (EA) extracts of propolis further decreased the zeta potential from -17 to -20 mV. Confocal laser scanning microscopy (CLSM) depicted that CPNP effectively disrupted biofilm formation by S. epidermidis and decreased viability to ~25% compared to Eth and EA with viability of ~60–70%. CPNP was more effective in reducing the viability of both planktonic as well as biofilm bacteria compared to Eth and EA. At 100 μg/mL concentration, CPNP decreased the survival of biofilm bacteria by ~70% compared to Eth or EA extracts which decreased viability by only 40%-50%. The morphology of bacterial biofilm examined by scanning electron microscopy depicted partial disruption of biofilm by Eth and EA extracts and significant disruption by CPNP reducing bacterial number in the biofilm by ~90%. Real time quantitative PCR analysis of gene expression in treated bacteria showed that genes involved in intercellular adhesion such as IcaABCD, embp and other related genes were significantly downregulated by CPNP. In addition to having a direct inhibitory effect on the survival of S. epidermidis, CPNP showed synergism with the antibiotics rifampicin, ciprofloxacin, vancomycin and doxycycline suggestive of effective treatment regimens. This would help decrease antibiotic treatment dose by at least 4-fold in combination therapies thereby opening up ways of tackling antibiotic resistance in bacteria.

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<![CDATA[TyrR is involved in the transcriptional regulation of biofilm formation and D-alanine catabolism in Azospirillum brasilense Sp7.]]> https://www.researchpad.co/article/5c6f1503d5eed0c48467ac9f

Azospirillum brasilense is one of the most studied species of diverse agronomic plants worldwide. The benefits conferred to plants inoculated with Azospirillum have been primarily attributed to its capacity to fix atmospheric nitrogen and synthesize phytohormones, especially indole-3-acetic acid (IAA). The principal pathway for IAA synthesis involves the intermediate metabolite indole pyruvic acid. Successful colonization of plants by Azospirillum species is fundamental to the ability of these bacteria to promote the beneficial effects observed in plants. Biofilm formation is an essential step in this process and involves interactions with the host plant. In this study, the tyrR gene was cloned, and the translated product was observed to exhibit homology to TyrR protein, a NtrC/NifA-type activator. Structural studies of TyrR identified three putative domains, including a domain containing binding sites for aromatic amino acids in the N-terminus, a central AAA+ ATPase domain, and a helix-turn-helix DNA binding motif domain in the C-terminus, which binds DNA sequences in promoter-operator regions. In addition, a bioinformatic analysis of promoter sequences in A. brasilense Sp7 genome revealed that putative promoters encompass one to three TyrR boxes in genes predicted to be regulated by TyrR. To gain insight into the phenotypes regulated by TyrR, a tyrR-deficient strain derived from A. brasilense Sp7, named A. brasilense 2116 and a complemented 2116 strain harboring a plasmid carrying the tyrR gene were constructed. The observed phenotypes indicated that the putative transcriptional regulator TyrR is involved in biofilm production and is responsible for regulating the utilization of D-alanine as carbon source. In addition, TyrR was observed to be absolutely required for transcriptional regulation of the gene dadA encoding a D-amino acid dehydrogenase. The data suggested that TyrR may play a major role in the regulation of genes encoding a glucosyl transferase, essential signaling proteins, and amino acids transporters.

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<![CDATA[Efficacy of liposomal amphotericin B and anidulafungin using an antifungal lock technique (ALT) for catheter-related Candida albicans and Candida glabrata infections in an experimental model]]> https://www.researchpad.co/article/5c75ac7dd5eed0c484d088b2

Objective

The aims of this study were as follows. First, we sought to compare the in vitro susceptibility of liposomal amphotericin B (LAmB) and anidulafungin on Candida albicans and Candida glabrata biofilms growing on silicone discs. Second, we sought to compare the activity of LAmB versus anidulafungin for the treatment of experimental catheter-related C. albicans and C. glabrata infections with the antifungal lock technique in a rabbit model.

Methods

Two C. albicans and two C. glabrata clinical strains were used. The minimum biofilm eradication concentration for 90% eradication (MBEC90) values were determined after 48h of treatment with LAmB and anidulafungin. Confocal microscopy was used to visualize the morphology and viability of yeasts growing in biofilms. Central venous catheters were inserted into New Zealand rabbits, which were inoculated of each strain of C. albicans and C. glabrata. Then, catheters were treated for 48h with saline or with antifungal lock technique using either LAmB (5mg/mL) or anidulafungin (3.33mg/mL).

Results

In vitro: anidulafungin showed greater activity than LAmB against C. albicans and C. glabrata strains. For C. albicans: MBEC90 of anidulafungin versus LAmB: CA176, 0.03 vs. 128 mg/L; CA180, 0.5 vs. 64 mg/L. For C. glabrata: MBEC90 of anidulafungin versus LAmB: CG171, 0.5 vs. 64 mg/L; CG334, 2 vs. 32 mg/L. In vivo: for C. albicans species, LAmB and anidulafungin achieved significant reductions relative to growth control of log10 cfu recovered from the catheter tips (CA176: 3.6±0.3 log10 CFU, p≤0.0001; CA180: 3.8±0.1 log10 CFU, p≤0.01). For C. glabrata, anidulafungin lock therapy achieved significant reductions relative to the other treatments (CG171: 4.8 log10 CFU, p≤0.0001; CG334: 5.1 log10 CFU, p≤0.0001)

Conclusions

For the C. albicans strains, both LAmB and anidulafungin may be promising antifungal lock technique for long-term catheter-related infections; however, anidulafungin showed significantly higher activity than LAmB against the C. glabrata strains.

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<![CDATA[Analysis of Pseudomonas aeruginosa biofilm membrane vesicles supports multiple mechanisms of biogenesis]]> https://www.researchpad.co/article/5c6f151dd5eed0c48467ade7

Outer Membrane Vesicles (OMVs) are ubiquitous in bacterial environments and enable interactions within and between species. OMVs are observed in lab-grown and environmental biofilms, but our understanding of their function comes primarily from planktonic studies. Planktonic OMVs assist in toxin delivery, cell-cell communication, horizontal gene transfer, small RNA trafficking, and immune system evasion. Previous studies reported differences in size and proteomic cargo between planktonic and agar plate biofilm OMVs, suggesting possible differences in function between OMV types. In Pseudomonas aeruginosa interstitial biofilms, extracellular vesicles were reported to arise through cell lysis, in contrast to planktonic OMV biogenesis that involves the Pseudomonas Quinolone Signal (PQS) without appreciable autolysis. Differences in biogenesis mechanism could provide a rationale for observed differences in OMV characteristics between systems. Using nanoparticle tracking, we found that P. aeruginosa PAO1 planktonic and biofilm OMVs had similar characteristics. However, P. aeruginosa PA14 OMVs were smaller, with planktonic OMVs also being smaller than their biofilm counterparts. Large differences in Staphylococcus killing ability were measured between OMVs from different strains, and a smaller within-strain difference was recorded between PA14 planktonic and biofilm OMVs. Across all conditions, the predatory ability of OMVs negatively correlated with their size. To address biogenesis mechanism, we analyzed vesicles from wild type and pqsA mutant biofilms. This showed that PQS is required for physiological-scale production of biofilm OMVs, and time-course analysis confirmed that PQS production precedes OMV production as it does in planktonic cultures. However, a small sub-population of vesicles was detected in pqsA mutant biofilms whose size distribution more resembled sonicated cell debris than wild type OMVs. These results support the idea that, while a small and unique population of vesicles in P. aeruginosa biofilms may result from cell lysis, the PQS-induced mechanism is required to generate the majority of OMVs produced by wild type communities.

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<![CDATA[C4-HSL aptamers for blocking qurom sensing and inhibiting biofilm formation in Pseudomonas aeruginosa and its structure prediction and analysis]]> https://www.researchpad.co/article/5c75abffd5eed0c484d07f98

This study aimed to screen DNA aptamers against the signal molecule C4-HSL of the rhl system for the inhibition of biofilm formation of Pseudomonas aeruginosa using an improved systematic evolution of ligand by exponential enrichment (SELEX) method based on a structure-switching fluorescent activating bead. The aptamers against the C4-HSL with a high affinity and specifity were successfully obtained and evaluated in real-time by this method. Results of biofilm inhibition experiments in vitro showed that the biofilm formation of P. aeruginosa was efficiently reduced to about 1/3 by the aptamers compared with that of the groups without the aptamers. Independent secondary structure simulation and computer-aided tertiary structure prediction (3dRNA) showed that the aptamers contained a highly conserved Y-shaped structural unit. Therefore, this study benefits the search for new methods for the detection and treatment of P. aeruginosa biofilm formation.

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<![CDATA[The 5’-nucleotidase S5nA is dispensable for evasion of phagocytosis and biofilm formation in Streptococcus pyogenes]]> https://www.researchpad.co/article/5c5ca309d5eed0c48441f036

5’-nucleotidases are widespread among all domains of life. The enzymes hydrolyze phosphate residues from nucleotides and nucleotide derivatives. In some pathobiontic bacteria, 5’-nucleotidases contribute to immune evasion by dephosphorylating adenosine mono-, di-, or tri-phosphates, thereby either decreasing the concentration of pro-inflammatory ATP or increasing the concentration of anti-inflammatory adenosine, both acting on purinergic receptors of phagocytic cells. The strict human pathogen Streptococcus pyogenes expresses a surface-associated 5’-nucleotidase (S5nA) under infection conditions that has previously been discussed as a potential virulence factor. Here we show that deletion of the S5nA gene does not significantly affect growth in human blood, evasion of phagocytosis by neutrophils, formation of biofilms and virulence in an infection model with larvae of the greater wax moth Galleria mellonella in S. pyogenes serotypes M6, M18 and M49. Hence, the surface-associated 5’-nucleotidase S5nA seems dispensable for evasion of phagocytosis and biofilm formation in S. pyogenes.

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<![CDATA[Structural basis of DSF recognition by its receptor RpfR and its regulatory interaction with the DSF synthase RpfF]]> https://www.researchpad.co/article/5c61e8f4d5eed0c48496f4de

The diffusible signal factors (DSFs) are a family of quorum-sensing autoinducers (AIs) produced and detected by numerous gram-negative bacteria. The DSF family AIs are fatty acids, differing in their acyl chain length, branching, and substitution but having in common a cis-2 double bond that is required for their activity. In both human and plant pathogens, DSFs regulate diverse phenotypes, including virulence factor expression, antibiotic resistance, and biofilm dispersal. Despite their widespread relevance to both human health and agriculture, the molecular basis of DSF recognition by their cellular receptors remained a mystery. Here, we report the first structure–function studies of the DSF receptor regulation of pathogenicity factor R (RpfR). We present the X-ray crystal structure of the RpfR DSF-binding domain in complex with the Burkholderia DSF (BDSF), which to our knowledge is the first structure of a DSF receptor in complex with its AI. To begin to understand the mechanistic role of the BDSF–RpfR contacts observed in the biologically important complex, we have also determined the X-ray crystal structure of the RpfR DSF-binding domain in complex with the inactive, saturated isomer of BDSF, dodecanoic acid (C12:0). In addition to these ligand–receptor complex structures, we report the discovery of a previously overlooked RpfR domain and show that it binds to and negatively regulates the DSF synthase regulation of pathogenicity factor F (RpfF). We have named this RpfR region the RpfF interaction (FI) domain, and we have determined its X-ray crystal structure alone and in complex with RpfF. These X-ray crystal structures, together with extensive complementary in vivo and in vitro functional studies, reveal the molecular basis of DSF recognition and the importance of the cis-2 double bond to DSF function. Finally, we show that throughout cellular growth, the production of BDSF by RpfF is post-translationally controlled by the RpfR N-terminal FI domain, affecting the cellular concentration of the bacterial second messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). Thus, in addition to describing the molecular basis for the binding and specificity of a DSF for its receptor, we describe a receptor–synthase interaction regulating bacterial quorum-sensing signaling and second messenger signal transduction.

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<![CDATA[Metagenomic quorum quenching enzymes affect biofilm formation of Candida albicans and Staphylococcus epidermidis]]> https://www.researchpad.co/article/5c58d622d5eed0c4840316f5

Biofilm formation in the clinical environment is of increasing concern since a significant part of human infections is associated, and caused by biofilm establishment of (opportunistic) pathogens, for instance Candida albicans and Staphylococcus epidermidis. The rapidly increasing number of antibiotic-resistant biofilms urgently requires the development of novel and effective strategies to prevent biofilm formation ideally targeting a wide range of infectious microorganisms. Both, synthesis of extracellular polymeric substances and quorum sensing are crucial for biofilm formation, and thus potential attractive targets to combat undesirable biofilms.We evaluated the ability of numerous recently identified metagenome-derived bacterial quorum quenching (QQ) proteins to inhibit biofilm formation of C. albicans and S. epidermidis. Here, proteins QQ-5 and QQ-7 interfered with the morphogenesis of C. albicans by inhibiting the yeast-to-hyphae transition, ultimately leading to impaired biofilm formation. Moreover, QQ5 and QQ-7 inhibited biofilm formation of S. epidermidis; in case of QQ7 most likely due to induced expression of the icaR gene encoding the repressor for polysaccharide intercellular adhesin (PIA) synthesis, the main determinant for staphylococcal biofilm formation. Our results indicate that QQ-5 and QQ-7 are attractive potential anti-biofilm agents in the prevention and treatment of C. albicans and S. epidermidis mono-species biofilms, and potentially promising anti-biofilm drugs in also combating multi-species infections.

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<![CDATA[The peptidoglycan and biofilm matrix of Staphylococcus epidermidis undergo structural changes when exposed to human platelets]]> https://www.researchpad.co/article/5c57e669d5eed0c484ef3082

Staphylococcus epidermidis is a bacterium frequently isolated from contaminated platelet concentrates (PCs), a blood product used to treat bleeding disorders in transfusion patients. PCs offer an accidental niche for colonization of S. epidermidis by forming biofilms and thus avoiding clearance by immune factors present in this milieu. Using biochemical and microscopy techniques, we investigated the structural changes of the peptidoglycan (PG) and the biofilm matrix of S. epidermidis biofilms formed in whole-blood derived PCs compared to biofilms grown in glucose-supplemented trypticase soy broth (TSBg). Both, the PG and the biofilm matrix are primary mechanisms of defense against environmental stress. Here we show that in PCs, the S. epidermidis biofilm matrix is mainly of a proteinaceous nature with extracellular DNA, in contrast to the predominant polysaccharide nature of the biofilm matrix formed in TSBg cultures. PG profile studies demonstrated that the PG of biofilm cells remodels during PC storage displaying fewer muropeptides variants than those observed in TSBg. The PG muropeptides contain two chemical modifications (amidation and O-acetylation) previously associated with resistance to antimicrobial agents by other staphylococci. Our study highlights two key structural features of S. epidermidis that are remodeled when exposed to human platelets and could be used as targets to reduce septic transfusions events.

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<![CDATA[Sugar and iron: Toward understanding the antibacterial effect of ciclopirox in Escherichia coli]]> https://www.researchpad.co/article/5c42438cd5eed0c4845e0573

New antibiotics are needed against antibiotic-resistant gram-negative bacteria. The repurposed antifungal drug, ciclopirox, equally blocks antibiotic-susceptible or multidrug-resistant Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae clinical isolates, indicating that it is not affected by existing resistance mechanisms. Toward understanding how ciclopirox blocks growth, we screened E. coli mutant strains and found that disruption of genes encoding products involved in galactose salvage, enterobacterial common antigen synthesis, and transport of the iron binding siderophore, enterobactin, lowered the minimum inhibitory concentration of ciclopirox needed to block growth of the mutant compared to the isogenic parent strain. We found that ciclopirox induced enterobactin production and that this effect is strongly affected by the deletion of the galactose salvage genes encoding UDP-galactose 4-epimerase, galE, or galactose-1-phosphate uridylyltransferase, galT. As disruption of ECA synthesis activates the regulation of capsular synthesis (Rcs) phosphorelay, which inhibits bacterial swarming and promotes biofilm development, we test whether ciclopirox prevents activation of the Rcs pathway. Sub-inhibitory concentrations of ciclopirox increased swarming of the E. coli laboratory K12 strain BW25113 but had widely varying effects on swarming or surface motility of clinical isolate E. coli, A. baumannii, and K. pneumoniae. There was no effect of ciclopirox on biofilm production, suggesting it does not target Rcs. Altogether, our data suggest ciclopirox-mediated alteration of lipopolysaccharides stimulates enterobactin production and affects bacterial swarming.

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<![CDATA[Matrix metalloprotease-1 inhibits and disrupts Enterococcus faecalis biofilms]]> https://www.researchpad.co/article/5c42436bd5eed0c4845e0123

Enterococcus faecalis is a major opportunistic pathogen that readily forms protective biofilms leading to chronic infections. Biofilms protect bacteria from detergent solutions, antimicrobial agents, environmental stress, and effectively make bacteria 10 to 1000-fold more resistant to antibiotic treatment. Extracellular proteins and polysaccharides are primary components of biofilms and play a key role in cell survival, microbial persistence, cellular interaction, and maturation of E. faecalis biofilms. Degradation of biofilm components by mammalian proteases is an effective antibiofilm strategy because proteases are known to degrade bacterial proteins leading to bacterial cell lysis and growth inhibition. Here, we show that human matrix metalloprotease-1 inhibits and disrupts E. faecalis biofilms. MMPs are cell-secreted zinc- and calcium-dependent proteases that degrade and regulate various structural components of the extracellular matrix. Human MMP1 is known to degrade type-1 collagen and can also cleave a wide range of substrates. We found that recombinant human MMP1 significantly inhibited and disrupted biofilms of vancomycin sensitive and vancomycin resistant E. faecalis strains. The mechanism of antibiofilm activity is speculated to be linked with bacterial growth inhibition and degradation of biofilm matrix proteins by MMP1. These findings suggest that human MMP1 can potentially be used as a potent antibiofilm agent against E. faecalis biofilms.

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<![CDATA[Attachment strength and on-farm die-off rate of Escherichia coli on watermelon surfaces]]> https://www.researchpad.co/article/5c3e4f8fd5eed0c484d76beb

Pre-harvest contamination of produce has been a major food safety focus. Insight into the behavior of enteric pathogens on produce in pre-harvest conditions will aid in developing pre-harvest and post-harvest risk management strategies. In this study, the attachment strength (SR) and die-off rate of E. coli on the surface of watermelon fruits and the efficacy of aqueous chlorine treatment against strongly attached E. coli population were investigated. Watermelon seedlings were transplanted into eighteen plots. Prior to harvesting, a cocktail of generic E. coli (ATCC 23716, 25922 and 11775) was inoculated on the surface of the watermelon fruits (n = 162) and the attachment strength (SR) values and the daily die-off rates were examined up to 6 days by attachment assay. After 120 h, watermelon samples were treated with aqueous chlorine (150 ppm free chlorine for 3 min). The SR value of the E. coli cells on watermelon surfaces significantly increased (P<0.05) from 0.04 to 0.99 in the first 24 h, which was primarily due to the decrease in loosely attached population, given that the population of strongly attached cells was constant. Thereafter, there was no significant change in SR values, up to 120 h. The daily die-off rate of E. coli ranged from -0.12 to 1.3 log CFU/cm2. The chlorine treatment reduced the E. coli level by 4.2 log CFU/cm2 (initial level 5.6 log CFU/cm2) and 0.62 log CFU/cm2 (initial level 1.8 log CFU/cm2), on the watermelons that had an attachment time of 30 min and 120 h respectively. Overall, our findings revealed that the population of E. coli on watermelon surfaces declined over time in an agricultural environment. Microbial contamination during pre-harvest stages may promote the formation of strongly attached cells on the produce surfaces, which could influence the efficacy of post-harvest washing and sanitation techniques.

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<![CDATA[Deletion of genes involved in the ketogluconate metabolism, Entner-Doudoroff pathway, and glucose dehydrogenase increase local and invasive virulence phenotypes in Streptococcus pneumoniae]]> https://www.researchpad.co/article/5c3e4f3cd5eed0c484d733f0

Streptococcus pneumoniae displays increased resistance to antibiotic therapy following biofilm formation. A genome-wide search revealed that SP 0320 and SP 0675 (respectively annotated as 5-keto-D-gluconate-5-reductase and glucose dehydrogenase) contain the highest degree of homology to CsgA of Myxococcus xanthus, a signaling factor that promotes cell aggregation and biofilm formation. Single and double SP 0320 and SP 0675 knockout mutants were created in strain BS72; however, no differences were observed in the biofilm-forming phenotypes of mutants compared to the wild type strain. Using the chinchilla model of otitis media and invasive disease, all three mutants exhibited greatly increased virulence compared to the wild type strain (increased pus formation, tympanic membrane rupture, mortality rates). The SP 0320 gene is located in an operon with SP 0317, SP 0318 and SP 0319, which we bioinformatically annotated as being part of the Entner-Doudoroff pathway. Deletion of SP 0317 also resulted in increased mortality in chinchillas; however, mutations in SP 0318 and SP 0319 did not alter the virulence of bacteria compared to the wild type strain. Complementing the SP 0317, SP 0320 and SP 0675 mutant strains reversed the virulence phenotype. We prepared recombinant SP 0317, SP 0318, SP 0320 and SP 0675 proteins and confirmed their functions. These data reveal that disruption of genes involved in the degradation of ketogluconate, the Entner-Doudoroff pathway, and glucose dehydrogenase significantly increase the virulence of bacteria in vivo; two hypothetical models involving virulence triggered by reduced in carbon-flux through the glycolytic pathways are presented.

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<![CDATA[Candida–streptococcal interactions in biofilm-associated oral diseases]]> https://www.researchpad.co/article/5c1c0b11d5eed0c484427391 ]]> <![CDATA[Deep-sea anthropogenic macrodebris harbours rich and diverse communities of bacteria and archaea]]> https://www.researchpad.co/article/5c0841d6d5eed0c484fcae0a

The deep sea is the largest biome on earth, and microbes dominate in biomass and abundance. Anthropogenic litter is now almost ubiquitous in this biome, and its deposition creates new habitats and environments, including for microbial assemblages. With the ever increasing accumulation of this debris, it is timely to identify and describe the bacterial and archaeal communities that are able to form biofilms on macrodebris in the deep sea. Using 16S rRNA gene high throughput sequencing, we show for the first time the composition of bacteria and archaea on macrodebris collected from the deep sea. Our data suggest differences in the microbial assemblage composition across litter of different materials including metal, rubber, glass, fabric and plastic. These results imply that anthropogenic macrodebris provide diverse habitats for bacterial and archaeal biofilms and each may harbour distinct microbial communities.

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