Sessile Legionella pneumophila is able to grow on surfaces and generate structured monospecies biofilms

Currently, models for studying Legionella pneumophila biofilm formation rely on multi-species biofilms with low reproducibility or on growth in rich medium, where planktonic growth is unavoidable. The present study describes a new medium adapted to the growth of L. pneumophila monospecies biofilms in vitro. A microplate model was used to test several media. After incubation for 6 days in a specific biofilm broth not supporting planktonic growth, biofilms consisted of 5.36 ± 0.40 log (cfu cm−2) or 5.34 ± 0.33 log (gu cm−2). The adhered population remained stable for up to 3 weeks after initial inoculation. In situ confocal microscope observations revealed a typical biofilm structure, comprising cell clusters ranging up to 300 μm in height. This model is adapted to growing monospecies L. pneumophila biofilms that are structurally different from biofilms formed in a rich medium. High reproducibility and the absence of other microbial species make this model useful for studying genes involved in biofilm formation

Microbial differences between dental plaque and historic dental calculus are related to oral biofilm maturation stage

Dental calculus, calcified oral plaque biofilm, contains microbial and host biomolecules that can be used to study historic microbiome communities and host responses. Dental calculus does not typically accumulate as much today as historically, and clinical oral microbiome research studies focus primarily on living dental plaque biofilm. However, plaque and calculus reflect different conditions of the oral biofilm, and the differences in microbial characteristics between the sample types have not yet been systematically explored. Here, we compare the microbial profiles of modern dental plaque, modern dental calculus, and historic dental calculus to establish expected differences between these substrates.- Background - Results -- Authentication of a preserved oral biofilm in calculus samples -- Dental calculus and plaque biofilm communities are distinct -- Health-associated communities of dental plaque and calculus are distinct -- Signatures of health and of disease are shared in modern and historic calculus samples -- Microbial community differences between health and disease in calculus are poorly resolved -- Absence of caries-specific microbial profiles in dental calculus -- Microbial co-exclusion patterns in plaque and calculus reflect biofilm maturity -- Microbial complexes in plaque and calculus -- Functional prediction in calculus is poorly predictive of health status -- Proteomic profiles of historic healthy site calculus -- Correlations between taxonomic, proteomic, and metabolomic profiles - Discussion - Conclusions - Materials and methods --Historic and modern calculus sample collection DNA extraction -- DNA library construction and high-throughput sequencing -- DNA sequence processing -- Genetic assessment of historic calculus sample preservation -- Genetic microbial taxonomic profiling -- Principal component analysis -- Assessment of differentially abundant taxa -- Sparse partial least squares-discriminant analysis -- Assessment of microbial co-exclusion patterns -- Gene functional categorization with SEED -- Proteomics -- Metabolomics -- Regularized canonical correlation analysi

RNAseq reveals hydrophobins that are involved in the adaptation of aspergillus nidulans to lignocellulose

Background Sugarcane is one of the world’s most profitable crops. Waste steam-exploded sugarcane bagasse (SEB) is a cheap, abundant, and renewable lignocellulosic feedstock for the next-generation biofuels. In nature, fungi seldom exist as planktonic cells, similar to those found in the nutrient-rich environment created within an industrial fermenter. Instead, fungi predominantly form biofilms that allow them to thrive in hostile environments. Results In turn, we adopted an RNA-sequencing approach to interrogate how the model fungus, Aspergillus nidulans, adapts to SEB, revealing the induction of carbon starvation responses and the lignocellulolytic machinery, in addition to morphological adaptations. Genetic analyses showed the importance of hydrophobins for growth on SEB. The major hydrophobin, RodA, was retained within the fungal biofilm on SEB fibres. The StuA transcription factor that regulates fungal morphology was up-regulated during growth on SEB and controlled hydrophobin gene induction. The absence of the RodA or DewC hydrophobins reduced biofilm formation. The loss of a RodA or a functional StuA reduced the retention of the hydrolytic enzymes within the vicinity of the fungus. Hence, hydrophobins promote biofilm formation on SEB, and may enhance lignocellulose utilisation via promoting a compact substrate-enzyme-fungus structure. Conclusion This novel study highlights the importance of hydrophobins to the formation of biofilms and the efficient deconstruction of lignocellulose

Factors contributing to biofilm formation of Yersinia enterocolitica : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology, Massey University, Palmerston North, New Zealand

Content removed from thesis due to copyright reasons: Wang H., Palmer J., & Flint S. (2015). A rapid method for the nonselective enumeration of Yersinia enterocolitica, a foodborne pathogen associated with pork. Meat Science, 113: 59–61. doi:10.1016/j.meatsci.2015.11.005; Wang H., Tay M., Palmer J., Flint S. (2016) Biofilm formation of Yersinia enterocolitica and its persistence following treatment with different sanitation agents. Food Control, 73, 433-437. doi:10.1016/j.foodcont.2016.08.033Biofilms of pathogenic bacteria are recognised as a threat to food safety. The aim of the present study was to investigate the potential of Yersinia enterocolitica to form biofilms in the pork processing environment and identify the resistance of these biofilms to sanitation. The biofilm formation by Y. enterocolitica was monitored at conditions simulating pork processing environment under daily cleaning routine using an impedance method established in this study. Results showed that Y. enterocolitica had the potential to form biofilm and become resistant to sanitation in a pork processing environment. An investigation into the factors influencing biofilm formation of Y. enterocolitica indicated that the Ca2+ ion increased the level of biofilm formation. In addition, the presence of the virulence plasmid pYV is essential for the biofilm Ca2+ response. Further analysis of the bacterial cell surface properties and extracellular polymeric substance (EPS) composition suggested that the pYV+ cell surfaces are more negatively charged and more hydrophobic than the pYV- cells although no significant difference was observed with the addition of Ca2+. The pYV+ cells appear to produce more exopolysaccharide than the pYV- cells regardless of Ca2+ concentration. Ca2+ was able to increase the yield of extracellular DNA while the presence of pYV appeared to be dispensable in terms of extracellular DNA release. Analysis of cell wall protein revealed one protein expressed in the pYV+ cells but absent in the pYV- cells

Contribution of epilithic diatoms to benthic−pelagic coupling in a temperate river

Water residence time in the middle course of rivers is often too short to allow substantial phytoplankton development, and primary production is essentially provided by benthic phototrophic biofilms. However, cells occurring in the water column might derive from biofilm microalgae, and, reciprocally, sedimenting microalgae could represent a continuous source of colonizers for benthic biofilms. A comparative study of biofilm and pelagic microphytic communities (with special focus on diatoms) was carried out over 15 mo in the Garonne River, France. Diatoms dominated both biofilm and pelagic microphytic communities. Typically benthic diatoms were found in high abundance in the water column, and their biomass in the water was correlated with their biomass in the biofilm, indicating the benthic origin of these cells. Variations in river discharge and temperature drove the temporal distribution of benthic and pelagic communities: under high flow mixing (winter) communities showed the greatest similarity, and during low flow (summer)they differed the most. Even during low flow, typical benthic species were observed in the water column, indicating that benthic−pelagic exchanges were not exclusively due to high water flow. Moreover, during low flow periods, planktonic diatoms typically settled within biofilms, presumably because of higher water residence times, and/or upstream reservoir flushing

Understanding aspects of alginate biosynthesis and regulation by Pseudomonas aeruginosa : a thesis presented in partial fulfilment of the requirements of the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand

Alginate is a medically and industrially important polymer produced by seaweeds and certain bacteria. The bacterium Pseudomonas aeruginosa over-produces alginate during cystic fibrosis lung infections, forming biofilms, making the infection difficult to treat. Bacteria make alginate using membrane spanning multi-protein complexes. Although alginate biosynthesis and regulation have been studied in detail, there are still major gaps in knowledge. In particular, the requirement of AlgL (a periplasmic alginate degrading enzyme) and role played by MucR (an inner membrane c-di-GMP modulator) are not well understood. Here I show that AlgL and MucR are not essential for alginate production during biofilm growth. My findings suggest that while catalytically active AlgL negatively affects alginate production, expressing catalytically inactive AlgL enhances alginate yields. Furthermore, preliminary data show AlgL is not required for the stability or functionality of the alginate biosynthesis complex, suggesting that it is a free periplasmic protein dispensable for alginate production. These findings support the prediction that the primary function of AlgL is to degrade misguided alginate from the periplasm. For MucR, I show for the first time that its sensor domain mediates nitrate-induced suppression of alginate biosynthesis. This appears to occur at multiple levels in a manner only partially dependent on c-di-GMP signaling. These results indicate that MucR is associated with the negative effect of nitrate (and possibly denitrification) on alginate production. On the basis of these results, I propose a combination of nitrate (or denitrification intermediates), exogenous lyases and antimicrobial agents could be used to eliminate established chronic biofilm infections. Furthermore, catalytically inactive AlgL and/or homologs of MucR with disabled sensor motifs could be harnessed in non-pathogenic bacteria for producing tailor-made alginates

Autogenic versus environmental control during development of river biofilm

In the natural environment, microbial community structure of river biofilm is controlled by biotic and abiotic factors. This study explored the capacity to manipulate the structure of microbial communities by modifying environmental conditions during the course of biofilm development. River epilithic biofilm was cultivated in situ on artificial substrates placed parallel to river water flow. Substrates were incubated for 3 and 5.5 weeks in river to allow natural biofilm development, at two sites with contrasting physico-chemical characteristics. The first site (Aurade´ , Gers, France) was located in an agricultural watershed basin and the second site (Larroque, Haute-Garonne, France) was located in a forested watershed basin. After 3 weeks of biofilm development, a subset of substrates was collected from one site and transplanted to the second site where they remained for 2.5 further weeks. Epilithic bacterial community structure (at 3 weeks from each site and at 5.5 weeks from biofilms with and without transplantation) was assessed using PCR-DGGE of 16S rDNA fragment. Biofilm biomass was estimated using ash free dry mass (AFDM). After 3 weeks of development, biofilms from the two sites exhibited comparable AFDM values (average of 1.4¡0.2 g.mx2). A difference between the two sites was observed after 5.5 weeks of development: AFDM decreased for biofilms from the agricultural watershed basin (from 1.4 to 0.18 g.mx2) as a consequence of grazing pressure (Bithynia), and increased for biofilms from the forested agricultural watershed (from 1.4 to 2.6 g.mx2). Microbial community analyses revealed a differentiated community structure between biofilms from the different sites and exhibited a change of microbial community structure after 5.5 weeks of biofilm development. These observations confirm a process of ecological succession in microbial communities. Changing the incubation site during biofilm development modified the trajectory of these ecological successions, suggesting that site characteristics mainly conditioned the structure of these microbial communities

Resistance of S. Aureus Atcc 25923, E. Coli 055k59 No. 3912/41 and P. Aeruginosa 27/99 to the Wash-disinfectant «Milkodez»

The aim of the work – the article presents the results of determining of the resistance of S. aureus ATCC 25923, E. coli 055K59 No. 3912/4 and P. aeruginosa 27/99 test cultures in planktonic form and in biofilm to our developed «Milkodez» acid detergent.Materials and methods. Microbial biofilms were grown on MPB in 5 cm disposable plastic Petri dishes. To determine the effect of disinfectants on microbial biofilms, 3 Petri dishes with biofilms of each of the test cultures were used. One of the Petri dishes served as control and she had for 15 minutes made 5 cm3 of saline NaCl solution, in the second – 5 cm3 of hot water (t=70±5 °C), and in the third – 5 cm3 of acidic detergent «Milkodez». Microbial biofilms were fixed for 10 min. 96º with ethyl alcohol for 10 min. were stained with a 0.1 % solution of crystalline violet, and the remnants of the unabsorbed paint were removed with phosphate buffer. The biofilm dye was extracted with 96º of ethyl alcohol, which was photocolometrically investigated at 570 nm to establish the density of the formed biofilms. The density of the formed microbial biofilms was considered low in optical density of the extract up to 0.5 units, average – from 0.5 to 1.0 units; and high – over 1.0 unitsThe resistance of planktonic forms of test cultures of microorganisms to disinfectants was determined in sterile tubes, which made 10 cm3 (t=70±5 °C) of 0.5 % of their working solutions and 0.1 cm3 (1 billion microbial bodies) of the standard test – cultures. The culture was maintained for 15 min. and made ten – fold plantings on IPA in Petri dishes.Incubation of mesophilic microorganisms was carried out in a thermostat at a temperature of 30 °C, and psychrophilic – 20 °C. After 48 hours the calculation of the growing colonies were carried out. The results were expressed in colony forming units (CFU).Results. Due to the impact on microbial biofilms formed by the test cultures of S. aureus ATCC 25923, E. coli 055K59 No. 3912/41 and P. aeruginosa 27/99 for 15 min. 0.5 % solution of acid detergent «Milkodez» the optical density of the solutions was respectively 0.64, 0.72, 0.45 units. The results obtained indicate that the melkodez caused a decrease in the biofilm–forming ability of S. aureus ATCC 25923 3.2 times, in E. coli 055K59 No. 3912/41 – 1,7 times and in P. aeruginosa 27/99 – 2.8 times, compared to control. However, the density of one – day microbial biofilms formed by S. aureus ATCC 25923 and E. coli 055K59 No. 3912/41 was medium, and P. aeruginosa 27/99 was low. It has been proven that the «Milkodez» acid detergent developed is more effective than the prototype «Hypracid», since it caused the death of 100 % of planktonic test cultures and the number of S. aureus ATCC 25923, E. coli 055K59 No. 3912/41 and P. aeruginosa 27/99 formed in the biofilm that survived after its application was 2.7, 3.2 and 1.4 times lower, respectively.Conclusions. It was found that the test cultures were able to form high – density biofilms, since the optical density of the extract in the control was in the range from 1.28 to 2.05 units, which is greater than 1.0 units. Acid wash detergent «Milkodez» for 15 minutes of exposure causes the formation of S. aureus ATCC 25923, E. coli 055K59 No. 3912/41 and P. aeruginosa 27/99 biofilms of low and medium density and reduces their biofilm capacity by 3.2, 1.7 and 2.8 times, respectively. Its use provides the death of 100 % of the planktonic forms of the test cultures under study and reduces their number in the biofilm by 2.7, 3.2 and 1,4 times more, respectively, compared to «Hypracid» detergent

Biocompatibility and antibacterial properties of zirconium nitride coating on titanium abutments: An in vitro study

Improving soft tissue attachment and reducing bacterial colonization on titanium abutments are key factors for the long-term maintenance of healthy soft and hard peri-implant tissues. This in vitro study was conducted to compare the biocompatibility and antibacterial activity of four different surfaces: uncoated Ti6Al4V, anodized, and coated with titanium nitride or zirconium nitride. Surface topography was investigated with a high-resolution system for measuring surface finishes. Human gingival fibroblast (HGF) adhesion and proliferation were examined using MTT assay, Scanning Electron Microscopy (SEM) imaging, immunofluorescence analysis and real-time PCR for selected target genes. The hemolysis and AMES tests were performed to assess the chemical compounds' blood compatibility and mutagenic potential, respectively. Antibacterial activity was tested against five bacterial strains isolated from the oral cavity (Streptococcus salivarius, S. sanguinis, S. mutans, S. sobrinus, S. oralis), and the percentage of dead bacteria was calculated. Roughness measurements confirmed a substantial similarity between the surfaces and their compatibility with clinical applications. MTT assay, SEM analysis and immunofluorescence staining showed adhesion and proliferation of HGFs cultured on all the examined surfaces. PCR confirmed that HGFs produced extracellular matrix components efficiently on all the surfaces. No hemolytic activity was detected, and the AMES test confirmed the surfaces' clinical safety. For all tested bacterial strains, biofilms grown on the zirconium nitride surface showed a higher percentage of dead bacteria than on the other disks. The titanium nitride surface inactivated bacterial biofilms, too, but to a lesser extent