Dairy biofilm: Bacterial community diversity assessment and impact of the Lactococcus lactis bio adhesion on biofilm growth

Biofilms are the most common mode of bacterial growth in nature. Their formation occurs on organic or inorganic solid surfaces in contact with a liquid, on gas-liquid and liquid-liquid boundaries as well. The aims of this study were, by combining cell enumeration, scanning electron microscopy and denaturing gel gradient electrophoresis (DGGE), to characterize the structural dynamics of dairy biofilm growth in the environments with a nutrient flow, and to evaluate the impact of adhesion of Lactococcus lactis on the biofilm community depending on the incubation time. Significantly higher values of biofilm volume and thickness were observed under dynamic conditions after 55 h. The populations of gram-positive bacteria and fungi exhibited a significantly higher biofilm organization after 2 days of cultivation than that of gram-negative bacteria. Also, results showed that Lc. lactis was able to adhere to silicone surface and the produced biofilm in which the number of adhered gram-positive and gram-negative bacteria decreased by nine orders of magnitude after 48 h of contact. This study constitutes a step ahead in developing the strategies to prevent microbial colonization by lactococcal protective biofilm.     

Bacillus cereus adhesion: Real time investigation of the effect on the chemistry of industrial stainless steel

The initial microorganism adhesion on substrate is an important step for the biofilm formation. The surface properties of the stainless steel and B. cereus were characterized by the sessile drop technique. Moreover, the physicochemical properties of surface adhesion and the impact of bio adhesion to the stainless steel were determined at different time of contact (2, 4, 7, 9 and 24 h). The results showed that the strain was hydrophilic (Giwi = 3.37 mJ/m²), whereas the substratum has hydrophobic character (Giwi = ?57.6 mJ/m²). Stainless steel surface presents a weak electron-donor character (γ^? = 4.1 mJ/m²) conversely to B. cereus that presents an important parameter (γ^? = 31.6 mJ/m²). The bio adhesion was investigated at different time of contact. The data analysis after 2 h, confirmed the adhesion of B. cereus with an amount of 10 cfu/cm² which increased to 1.210⁴ cfu/cm² after 24 h. Interestingly, despite the difference of hydropohbicity, the interaction between B. cereus and substratum was favored by the thermodynamic aspect of adhesion (ΔG_adhesion < 0). Interestingly, the study of the effect of B. cereus adhesion on the stainless steel has revealed that, the substratum becomes hydrophilic (θ° = 41.3, ΔGiwi = 39.6 mJ/m²) and highly electron donor (Γ^? = 52.9 mJ/m²) after 2 h of bio adhesion.     

Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus: a chronicle of evolution in action

Background

Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus are lactic acid producing bacteria that are largely used in dairy industries, notably in cheese-making and yogurt production. An earlier in-depth study of the first completely sequenced ssp. bulgaricus genome revealed the characteristics of a genome in an active phase of rapid evolution, in what appears to be an adaptation to the milk environment. Here we examine for the first time if the same conclusions apply to the ssp. lactis, and discuss intra- and inter-subspecies genomic diversity in the context of evolutionary adaptation.

Results

Both L. delbrueckii ssp. show the signs of reductive evolution through the elimination of superfluous genes, thereby limiting their carbohydrate metabolic capacities and amino acid biosynthesis potential. In the ssp. lactis this reductive evolution has gone less far than in the ssp. bulgaricus. Consequently, the ssp. lactis retained more extended carbohydrate metabolizing capabilities than the ssp. bulgaricus but, due to high intra-subspecies diversity, very few carbohydrate substrates, if any, allow a reliable distinction of the two ssp. We further show that one of the most important traits, lactose fermentation, of one of the economically most important dairy bacteria, L. delbruecki ssp. bulgaricus, relies on horizontally acquired rather than deep ancestral genes. In this sense this bacterium may thus be regarded as a natural GMO avant la lettre.

Conclusions

The dairy lactic acid producing bacteria L. delbrueckii ssp. lactis and ssp. bulgaricus appear to represent different points on the same evolutionary track of adaptation to the milk environment through the loss of superfluous functions and the acquisition of functions that allow an optimized utilization of milk resources, where the ssp. bulgaricus has progressed further away from the common ancestor.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-407) contains supplementary material, which is available to authorized users.     

Occurrence of Staphylococcus aureus and evaluation of anti-staphylococcal activity of Lactococcus lactis subsp. lactis in ready-to-eat poultry meat

A total of 181 ready-to-eat poultry meat samples were examined for the presence of Staphylococcus aureus, and 11 (6 %) were found to have S. aureus contamination. Of 11 S. aureus isolates, 10 (91 %) were resistant to at least one antibiotic used in this study, and 2 were resistant to oxacillin. Lactococcus lactis subsp. lactis was tested as a bio-control agent. All the S. aureus isolates were found to be sensitive to antimicrobial products in L. lactis subsp. lactis supernatants; the zones of inhibition were in the range of 5.0 mm?±?0.70 mm to 19.8 mm?±?0.83 mm with the majority of isolates. As a competitive flora in mixed culture (LAPTg broth) and protective culture in poultry meat, L. lactis subsp. lactis was effective against S. aureus isolates; the growth of S. aureus isolates was almost negative after 32 h incubation in mixed culture. The population of S. aureus was reduced substantially to almost log 1 CFU/g after 25 days of incubation in protective culture. The pH of the test cultures also decreased sharply with time.     

Production of human lysozyme in biofilm reactor and optimization of growth parameters of Kluyveromyces lactis K7

Lysozyme (1,4-β-N-acetylmuramidase) is a lytic enzyme, which degrades the bacterial cell wall. Lysozyme has been of interest in medicine, cosmetics, and food industries because of its anti-bactericidal effect. Kluyveromyces lactis K7 is a genetically modified organism that expresses human lysozyme. There is a need to improve the human lysozyme production by K. lactis K7 to make the human lysozyme more affordable. Biofilm reactor provides high biomass by including a solid support, which microorganisms grow around and within. Therefore, the aim of this study was to produce the human lysozyme in biofilm reactor and optimize the growth conditions of K. lactis K7 for the human lysozyme production in biofilm reactor with plastic composite support (PCS). The PCS, which includes polypropylene, soybean hull, soybean flour, bovine albumin, and salts, was selected based on biofilm formation on PCS (CFU/g), human lysozyme production (U/ml), and absorption of lysozyme inside the support. To find the optimum combination of growth parameters, a three-factor Box–Behnken design of response surface method was used. The results suggested that the optimum conditions for biomass and lysozyme productions were different (27 °C, pH 6, 1.33 vvm for biomass production; 25 °C, pH 4, no aeration for lysozyme production). Then, different pH and aeration shift strategies were tested to increase the biomass at the first step and then secrete the lysozyme after the shift. As a result, the lysozyme production amount (141 U/ml) at 25 °C without pH and aeration control was significantly higher than the lysozyme amount at evaluated pH and aeration shift conditions (p?<?0.05).     

Microbial Treatment for Prevention and Removal of Paraffin Deposition on the Walls of Crude Pipelines

Two bacterial strains, paraffin removal strain and biosurfactant-producing strain, named BHJ-1 and QFL-1, were isolated from oil production wells in Daqing oilfield of China. They were subsequently identified as Bacillus cereus QAU68 and Bacillus subtilis XCCX, respectively. As an indicator of the degradation paraffin, the inoculum concentration of BHJ-1 and QFL-1 were added in different proportions, the optimum proportion was 5:2. In this proportion the degradation rate of paraffin could reach 64 %, the prevention rate of paraffin could reach 55 %.     

Development of a PCR-RFLP assay for the identification of Lactococcus lactis ssp. lactis and cremoris

The development of new starter culture of Lactococcus lactis for the manufacture of fermented dairy products with unique characteristics usually requires the isolation and identification of L. lactis up to subspecies level. Therefore, a rapid and specific PCR-RFLP assay has been developed. Forward and reverse primer sets were designed targeting the conserved house keeping gene htrA and yueF encoding a trypsin-like serine protease and a non-proteolytic protein from peptidase family M16, respectively, of L. lactis. Amplicons of 265 bp and 447 bp of htrA and yueF, respectively, were subjected to restriction fragment length polymorphism analysis. Restriction of the 265 bp amplicons with TaqI produced DNA bands of 90 bp and 175 bp with ssp. lactis, and 66 bp and 199 bp with ssp. cremoris. Similarly, restriction of PCR product of 447 bp size with AluI produced digested fragments of 125 bp and 322 bp with ssp. lactis, and 71 bp and 376 bp with ssp. cremoris. The designed primer sets were observed to be specific to L. lactis because other bacteria could not be amplified. The ssp. lactis and cremoris of L. lactis could be identified by restriction of PCR products of htrA and yueF with TaqI and AluI, respectively.     

Impact of the exopolysaccharide layer on biofilms,adhesion and resistance to stress in Lactobacillus johnsonii FI9785

Background

The bacterial cell surface is a crucial factor in cell-cell and cell-host interactions. Lactobacillus johnsonii FI9785 produces an exopolysaccharide (EPS) layer whose quantity and composition is altered in mutants that harbour genetic changes in their eps gene clusters. We have assessed the effect of changes in EPS production on cell surface characteristics that may affect the ability of L. johnsonii to colonise the poultry host and exclude pathogens.

Results

Analysis of physicochemical cell surface characteristics reflected by Zeta potential and adhesion to hexadecane showed that an increase in EPS gave a less negative, more hydrophilic surface and reduced autoaggregation. Autoaggregation was significantly higher in mutants that have reduced EPS, indicating that EPS can mask surface structures responsible for cell-cell interactions. EPS also affected biofilm formation, but here the quantity of EPS produced was not the only determinant. A reduction in EPS production increased bacterial adhesion to chicken gut explants, but made the bacteria less able to survive some stresses.

Conclusions

This study showed that manipulation of EPS production in L. johnsonii FI9785 can affect properties which may improve its performance as a competitive exclusion agent, but that positive changes in adhesion may be compromised by a reduction in the ability to survive stress.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0347-2) contains supplementary material, which is available to authorized users.     

In Vitro Characterization of a Recombinant AHL-Lactonase from Bacillus cereus Isolated from a Striped Catfish (Pangasianodon hypophthalmus) Pond

aiiA gene encoding AHL-lactonase was isolated from Bacillus cereus strain N26.2, originating from a striped catfish (Pangasianodon hypophthalmus) pond in Vietnam. This gene, abbreviated as aiiA(N26.2), was cloned and expressed in a competent Escherichia coli strain BL21(DE3)pLysS. The resulting protein, abbreviated as AiiA_N26.2, was highly active in the pH range of 6–8 and could retain 80 % of the maximum activity under storage for 5 days at 4 °C or for 3 days at 20 °C. These properties of AiiA_N26.2 protein confers its future application via feed supplementation, with the purpose of controlling aquaculture pathogens which regulate the virulence via a quorum sensing system.     

A flow chamber assay for quantitative evaluation of bacterial surface colonization used to investigate the influence of temperature and surface hydrophilicity on the biofilm forming capacity of uropathogenic Escherichia coli

We have established a simple flow chamber-based procedure which provides an accurate and reproducible way to measure the amount of biofilm formed on an implantable biomaterial surface. The method enables the side-by-side evaluation of different materials under hydrodynamic flow conditions similar to those found on an implanted device. We have used the method to evaluate the biofilm forming capacity of clinically isolated Escherichia coli on silicone rubber and on silicone rubber containing a hydrophilic coating. It was found that the surface chemistry influenced the colonization of the isolates very differently. In addition, the temperature was found to have a considerable influence upon the adhesion and biofilm forming capacity of some of the isolates, and that the influence of surface chemistry depended on temperature. Our results suggest that the step from using E. coli laboratory strains to clinical isolates entails a significant rise in complexity and yields results that cannot be generalized. The results should be valuable information for researchers working with pre-clinical evaluation of device-associated E. coli infections.     

Genome-wide metabolic re-annotation of Ashbya gossypii: new insights into its metabolism through a comparative analysis with Saccharomyces cerevisiae and Kluyveromyces lactis

Background

Ashbya gossypii is an industrially relevant microorganism traditionally used for riboflavin production. Despite the high gene homology and gene order conservation comparatively with Saccharomyces cerevisiae, it presents a lower level of genomic complexity. Its type of growth, placing it among filamentous fungi, questions how close it really is from the budding yeast, namely in terms of metabolism, therefore raising the need for an extensive and thorough study of its entire metabolism. This work reports the first manual enzymatic genome-wide re-annotation of A. gossypii as well as the first annotation of membrane transport proteins.

Results

After applying a developed enzymatic re-annotation pipeline, 847 genes were assigned with metabolic functions. Comparatively to KEGG’s annotation, these data corrected the function for 14% of the common genes and increased the information for 52 genes, either completing existing partial EC numbers or adding new ones. Furthermore, 22 unreported enzymatic functions were found, corresponding to a significant increase in the knowledge of the metabolism of this organism. The information retrieved from the metabolic re-annotation and transport annotation was used for a comprehensive analysis of A. gossypii’s metabolism in comparison to the one of S. cerevisiae (post-WGD – whole genome duplication) and Kluyveromyces lactis (pre-WGD), suggesting some relevant differences in several parts of their metabolism, with the majority being found for the metabolism of purines, pyrimidines, nitrogen and lipids. A considerable number of enzymes were found exclusively in A. gossypii comparatively with K. lactis (90) and S. cerevisiae (13). In a similar way, 176 and 123 enzymatic functions were absent on A. gossypii comparatively to K. lactis and S. cerevisiae, respectively, confirming some of the well-known phenotypes of this organism.

Conclusions

This high quality metabolic re-annotation, together with the first membrane transporters annotation and the metabolic comparative analysis, represents a new important tool for the study and better understanding of A. gossypii’s metabolism.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-810) contains supplementary material, which is available to authorized users.     

Effective conversion of industrial starch waste to L-Lactic acid by Lactococcus lactis in a dialysis sac bioreactor

We describe here a simple technological process based on the direct fermentation of potato starch waste (PSW), an inexpensive agro-processing industrial waste, by a potential probiotic strain, Lactococcus lactis subsp. lactis, for enhancing L-lactic acid production. To maximize bioconversion and increase cell stability, we designed and tested a novel dialysis sac-based bioreactor. Shake flask fermentation (SFF) and fed batch fermentation in the dialysis sac bioreactor were compared for L-lactic acid production efficiency. The results showed that the starch (20 g/L) in the PSW-containing medium was completely consumed within 24 h in the dialysis sac bioreactor, compared with 48 h in the SFF. The maximum lactic acid concentration (18.9 g/L) and lactic acid productivity (0.79 g/L·h) obtained was 1.2- and 2.4-fold higher in the bioreactor than by SFF, respectively. Simultaneous saccharification and fermentation was effected at pH 5.5 and 30 °C. L. lactis cells were viable for up to four cycles in the fed batch fermentation compared to only one cycle in the SFF.     

Conjugal transfer between Bacillus thuringiensis and Bacillus cereus strains is not directly correlated with growth of recipient strains

Bacillus thuringiensis and Bacillus cereus belong to the B. cereus species group. The two species share substantial chromosomal similarity and differ mostly in their plasmid content. The phylogenetic relationship between these species remains a matter of debate. There is genetic exchange both within and between these species, and current evidence indicates that insects are a particularly suitable environment for the growth of and genetic exchange between these species. We investigated the conjugation efficiency of B. thuringiensis var. kurstaki KT0 (pHT73-Em^R) as a donor and a B. thuringiensis and several B. cereus strains as recipients; we used one-recipient and two-recipient conjugal transfer systems in vitro (broth and filter) and in Bombyx mori larvae, and assessed multiplication following conjugation between Bacillus strains. The B. thuringiensis KT0 strain did not show preference for genetic exchange with the B. thuringiensis recipient strain over that with the B. cereus recipient strains. However, B. thuringiensis strains germinated and multiplied more efficiently than B. cereus strains in insect larvae and only B. thuringiensis maintained complete spore germination for at least 24 h in B. mori larvae. These findings show that there is no positive association between bacterial multiplication efficiency and conjugation ability in infected insects for the used strains.     

Effect of Ottoman Viper (Montivipera xanthina (Gray, 1849)) Venom on Various Cancer Cells and on Microorganisms

Cytotoxic and antimicrobial effects of Montivipera xanthina venom against LNCaP, MCF-7, HT-29, Saos-2, Hep3B, Vero cells and antimicrobial activity against selected bacterial and fungal species: Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, E. coli O157H7, Enterococcus faecalis 29212, Enterococcus faecium DSM 13590, Staphylococcus epidermidis ATCC 12228, S. typhimirium CCM 5445, Proteus vulgaris ATCC 6957 and Candida albicans ATCC 10239 were studied for evaluating the potential medical benefit of this snake venom. Cytotoxicity of venom was determined using MTT assay. Snake venom cytotoxicity was expressed as the venom dose that killed 50 % of the cells (IC₅₀). The antimicrobial activity of venom was studied by minimal inhibitory concentration (MIC) and disc diffusion assay. MIC was determined using broth dilution method. The estimated IC₅₀ values of venom varied from 3.8 to 12.7 or from 1.9 to 7.2 μg/ml after treatment with crude venom for 24 or 48 h for LNCaP, MCF-7, HT-29 and Saos-2 cells. There was no observable cytotoxic effect on Hep3B and Vero cells. Venom exhibited the most potent activity against C. albicans (MIC, 7.8 μg/ml and minimal fungicidal concentration, 62.5 μg/ml) and S. aureus (MIC, 31.25 μg/ml). This study is the first report showing the potential of M. xanthina venom as an alternative therapeutic approach due to its cytotoxic and antimicrobial effects.     

Influence of inoculation with plant growth promoting rhizobacteria (PGPR) on tomato plant growth and nematode reproduction under greenhouse conditions

Numerous species of soil bacteria which flourish in the rhizosphere of plants or around plant tissues stimulate plant growth and reduce nematode population by antagonistic behavior. These bacteria are collectively known as PGPR (plant growth promoting rhizobacteria). The effects of six isolates of PGPR Pseudomonas putida, Pseudomonas fluorescens, Serratia marcescens, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus cereus, were studied on tomato plant growth and root knot nematode reproduction after 45 days from nematode infection. The highest number of shoot dry weight/g (43.00 g) was detected in the plant treated with S. marcescens; then P. putida (34.33 g), B. amyloliquefaciens (31.66 g), P. fluorescens (30.0 g), B. subtilis (29.0 g), B. cereus (27.0 g) and nematode alone (untreated) 20 g/plant. While the highest number of plant height was observed when plant was treated with S. marcescens, P. fluorescens, P. putida, B. amyloliquefaciens and P. putida 52.66, 50.66, 48 and 48 cm respectively. No significant differences were seen between previous treatments but only had significant differences compared with untreated plant. The highest number of fruit/plant was observed when plants were treated with S. marcescens (10.66), then B. amyloliquefaciens (8.66), P. putida (8), P. fluorescens (8) and B. cereus (7.66). No significant differences between the last 4 treatments, but all had significant differences compared with untreated plants. The highest weight of plant yield (g) was observed with S. marcescens (319.6 g/plant) and the lowest weight of plant yield was observed in plants treated with nematode alone (untreated). On the other hand, the lowest numbers of J₂/10 g of soil (78), galls/root, (24.33) galls/root, egg masses/root (12.66) and egg/egg masses were observed in the plants treated with S. marcescens.     

Purification and Characterization of Bacteriocin Produced by Lactobacillus brevis UN Isolated from Dhulliachar: a Traditional Food Product of North East India

A bacteriocin producing strain Lactobacillus brevis UN isolated from Dulliachar—a salted pickle and identified by biochemical and molecular methods. L. brevis UN was found to produce bacteriocin with broad spectrum activity against spoilage causing/food borne pathogens viz. L. monocytogenes, C. perfringens, S. aureus, L. mesenteroides, L. plantarum and B. cereus. Bacteriocin production was optimized through classical one variable at a time method. The isolate showed maximum bacteriocin production at early stationary phase, pH 4.0, temperature 35 °C and with an inoculum size of 1.5 OD @ 10 %. Bacteriocin produced by L. brevis UN was purified to homogeneity by single step gel exclusion chromatography and was most active at pH 6.0 and 7.0, stable up to 100 °C and was proteinaceous in nature. The results of NMR revealed the presence of proline, glutamic acid, aspartic acid, leucine, isoleucine and serine in its peptide structure. PCR amplification analysis determined that bacteriocin encoded gene in L. brevis UN was plasmid bound.     

Adhesion of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> biofilms to glass,stainless steel and cellulose

Objectives

The adhesion of colloidal probes of stainless steel, glass and cellulose to Pseudomonas fluorescens biofilms was examined using atomic force microscopy (AFM) to allow comparisons between surfaces to which biofilms might adhere.

Results

Biofilm was grown on a stainless steel substrate and covered most of the surface after 96 h. AFM approach and retraction curves were obtained when the biofilm was immersed in a tryptone/soy medium. On approach, all the colloidal probes experienced a long non-contact phase more than 100 nm in length, possibly due to the steric repulsion by extracellular polymers from the biofilm and hydrophobic effects. Retraction data showed that the adhesion varied from position to position on the biofilm. The mean value of adhesion of glass to the biofilm (48 ± 7 nN) was the greatest, followed by stainless steel (30 ± 7 nN) and cellulose (7.8 ± 0.4 nN).

Conclusion

The method allows understanding of adhesion between the three materials and biofilm, and development of a better strategy to remove the biofilm from these surfaces relevant to different industrial applications.

     

The effects of glutaraldehyde on the control of single and dual biofilms of Bacillus cereus and Pseudomonas fluorescens

Glutaraldehyde (GLUT) was evaluated for control of single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens on stainless steel surfaces using a chemostat system. The biofilms were characterized in terms of mass, cell density, total and matrix proteins and polysaccharides. The control action of GLUT was assessed in terms of inactivation and removal of biofilm. Post-biocide action was characterized 3, 7, 12, 24, 48 and 72 h after treatment. Tests with planktonic cells were also performed for comparison. The results demonstrated that in dual species biofilms the metabolic activity, cell density and the content of matrix proteins were higher than those of either single species. Planktonic B. cereus was more susceptible to GLUT than P. fluorescens. The biocide susceptibility of dual species planktonic cultures was an average of each single species. Planktonic cells were more susceptible to GLUT than their biofilm counterparts. Biofilm inactivation was similar for both of the single biofilms while dual biofilms were more resistant than single species biofilms. GLUT at 200 mg l^?1 caused low biofilm removal (<10%). Analysis of the post-biocide treatment data revealed the ability of biofilms to recover their activity over time. However, 12 h after biocide application, sloughing events were detected for both single and dual species biofilms, but were more marked for those formed by P. fluorescens (removal >40% of the total biofilm). The overall results suggest that GLUT exerts significant antimicrobial activity against planktonic bacteria and a partial and reversible activity against B. cereus and P. fluorescens single and dual species biofilms. The biocide had low antifouling effects when analysed immediately after treatment. However, GLUT had significant long-term effects on biofilm removal, inducing significant sloughing events (recovery in terms of mass 72 h after treatment for single biofilms and 42 h later for dual biofilms). In general, dual species biofilms demonstrated higher resistance and resilience to GLUT exposure than either of the single species biofilms. P. fluorescens biofilms were more susceptible to the biocide than B. cereus biofilms.