Biomineralization of manganese by Bacillus spp isolated from a marine biofilm

Biomineralization of manganese on titanium condenser material exposed to seawater has been illustrated. Biomineralization occurs when the fouling components, namely, the microbes, are able to oxidize minerals present in water and deposit them as insoluble oxides on biofilm surfaces. Extensive biofilm characterization studies Showed that an alarmingly large number of bacteria in these biofilms are capable of oxidizing manganese and are, thereby, capable of causing biomineralization on the condenser material exposed to seawater. This paper addresses studies on understanding the exact role of the microbes in bringing about oxidation of manganese. The kinetics of manganese oxidation by marine Gram-positive manganese oxidizing bacterium Bacillus spp. that was isolated front the titanium surface was studied in detail. Manganese oxidation in the presence of Bacillus cells, by cell free extract (CFE) and heat-treated cell free extract was also studied. The study confirmed that bacteria mediate manganese oxidation and lead to the formation of biogenic oxides of MnO2 eventually leading to biomineralization on titanium surface exposed to seawater.

Proteomics and mass spectrometric studies reveal planktonic growth of Mycobacterium smegmatis in biofilm cultures in the absence of rpoZ

Mycobacterium smegmatis is known to form biofilms and many cell surface molecules like core glycopeptidolipids and short-chain mycolates appear to play important role in the process. However, the involvement of the cell surface molecules in mycobacteria towards complete maturation of biofilms is still not clear. This work demonstrates the importance of the glycopeptidolipid species with hydroxylated alkyl chain and the epoxylated mycolic acids, during the process of biofilm development. In our previous study, we reported the impairment of biofilm formation in rpoZ-deleted M. smegmatis, where rpoZ codes for the ω subunit of RNA polymerase (R. Mathew, R. Mukherjee, R. Balachandar, D. Chatterji, Microbiology 152 (2006) 1741). Here we report the occurrence of planktonic growth in a mc2155 strain which is devoid of rpoZ gene. This strain is deficient in selective incorporation of the hydroxylated glycopeptidolipids and the epoxy mycolates to their respective locations in the cell wall. Hence it forms a mutant biofilm defective in maturation, wherein the cells undertake various alternative metabolic pathways to survive in an environment where oxygen, the terminal electron acceptor, is limiting.

Regulation of lipid biosynthesis, sliding motility, and biofilm formation by a membrane-anchored nucleoid-associated protein of mycobacterium tuberculosis

Bacteria use a number of small basic proteins for organization and compaction of their genomes. By their interaction with DNA, these nucleoid-associated proteins (NAPs) also influence gene expression. Rv3852, a NAP of Mycobacterium tuberculosis, is conserved among the pathogenic and slow-growing species of mycobacteria. Here, we show that the protein predominantly localizes in the cell membrane and that the carboxy-terminal region with the propensity to form a transmembrane helix is necessary for its membrane localization. The protein is involved in genome organization, and its ectopic expression in Mycobacterium smegmatis resulted in altered nucleoid morphology, defects in biofilm formation, sliding motility, and change in apolar lipid profile. We demonstrate its crucial role in regulating the expression of KasA, KasB, and GroEL1 proteins, which are in turn involved in controlling the surface phenotypes in mycobacteria.

Antimicrobial Peptides with Potential for Biofilm Eradication: Synthesis and Structure Activity Relationship Studies of Battacin Peptides

We report on the first chemical syntheses and structureactivity analyses of the cyclic lipopeptide battacin which revealed that conjugation of a shorter fatty acid, 4-methyl-hexanoic acid, and linearization of the peptide sequence improves antibacterial activity and reduces hemolysis of mouse blood cells. This surprising finding of higher potency in linear lipopeptides than their cyclic counterparts is economically beneficial. This novel lipopeptide was membrane lytic and exhibited antibiofilm activity against Pseudomonas aeruginosa, Staphylococcus aureus, and, for the first time, Pseudomonas syringe pv. actinidiae. The peptide was unstructured in aqueous buffer and dimyristoylphosphatidylcholine-polymerized diacetylene vesicles, with 12% helicity induced in 50% v/v of trifluoroethanol. Our results indicate that a well-defined secondary structure is not essential for the observed antibacterial activity of this novel lipopeptide. A truncated pentapeptide conjugated to 4-methyl hexanoic acid, having similar potency against Gram negative and Gram positive pathogens was identified through alanine scanning.

Quorum Sensing and Biofilm Formation in Mycobacteria: Role of c-di-GMP and Methods to Study This Second Messenger

Bacteria have evolved to survive the ever-changing environment using intriguing mechanisms of quorum sensing (QS). Very often, QS facilitates formation of biofilm to help bacteria to persist longer and the formation of such biofilms is regulated by c-di-GMP. It is a well-known second messenger also found in mycobacteria. Several methods have been developed to study c-di-GMP signaling pathways in a variety of bacteria. In this review, we have attempted to highlight a connection between c-di-GMP and biofilm formation and QS in mycobacteria and several methods that have helped in better understanding of c-di-GMP signaling. (c) 2014 IUBMB Life, 66(12):823-834, 2014

Chemical Functionalization of Graphene To Augment Stem Cell Osteogenesis and Inhibit Biofilm Formation on Polymer Composites for Orthopedic Applications

Toward designing the next generation of resorbable biomaterials for orthopedic applications, we studied poly(epsilon-caprolactone) (PCL) composites containing graphene. The role, if any, of the functionalization of graphene on mechanical properties, stem cell response, and biofilm formation was systematically evaluated. PCL composites of graphene oxide (GO), reduced GO (RGO), and amine-functionalized GO (AGO) were prepared at different filler contents (1%, 3%, and 5%). Although the addition of the nanoparticles to PCL markedly increased the storage modulus, this increase was largest for GO followed by AGO and RGO. In vitro cell studies revealed that the AGO and GO particles significantly increased human mesenchymal stem cell proliferation. AGO was most effective in augmenting stem cell osteogenesis leading to mineralization. Bacterial studies revealed that interaction with functionalized GO induced bacterial cell death because of membrane damage, which was further accentuated by amine groups in AGO. As a result, AGO composites were best at inhibiting biofilm formation. The synergistic effect of oxygen containing functional groups and amine groups on AGO imparts the optimal combination of improved modulus, favorable stem cell response, and biofilm inhibition in AGO-reinforced composites desired for orthopedic applications. This work elucidates the importance of chemical functionalization of graphene in polymer composites for biomedical applications.

Cytotoxicity of Ultrasmall Gold Nanoparticles on Planktonic and Biofilm Encapsulated Gram-Positive Staphylococci

The emergence of multidrug resistant bacteria, especially biofilm-associated Staphylococci, urgently requires novel antimicrobial agents. The antibacterial activity of ultrasmall gold nanoparticles (AuNPs) is tested against two gram positive: S. aureus and S. epidermidis and two gram negative: Escherichia coli and Pseudomonas aeruginosa strains. Ultrasmall AuNPs with core diameters of 0.8 and 1.4 nm and a triphenylphosphine-monosulfonate shell (Au0.8MS and Au1.4MS) both have minimum inhibitory concentration (MIC) and minimum bactericidal concentration of 25 x 10(-6)m Au]. Disc agar diffusion test demonstrates greater bactericidal activity of the Au0.8MS nanoparticles over Au1.4MS. In contrast, thiol-stabilized AuNPs with a diameter of 1.9 nm (AuroVist) cause no significant toxicity in any of the bacterial strains. Ultrasmall AuNPs cause a near 5 log bacterial growth reduction in the first 5 h of exposure, and incomplete recovery after 21 h. Bacteria show marked membrane blebbing and lysis in biofilm-associated bacteria treated with ultrasmall AuNP. Importantly, a twofold MIC dosage of Au0.8MS and Au1.4MS each cause around 80%-90% reduction in the viability of Staphylococci enveloped in biofilms. Altogether, this study demonstrates potential therapeutic activity of ultrasmall AuNPs as an effective treatment option against staphylococcal infections.

Chronic lung infection by Pseudomonas aeruginosa biofilm is cured by L-Methionine in combination with antibiotic therapy

Bacterial biofilms are associated with 80-90% of infections. Within the biofilm, bacteria are refractile to antibiotics, requiring concentrations >1,000 times the minimum inhibitory concentration. Proteins, carbohydrates and DNA are the major components of biofilm matrix. Pseudomonas aeruginosa (PA) biofilms, which are majorly associated with chronic lung infection, contain extracellular DNA (eDNA) as a major component. Herein, we report for the first time that L-Methionine (L-Met) at 0.5 mu M inhibits Pseudomonas aeruginosa (PA) biofilm formation and disassembles established PA biofilm by inducing DNase expression. Four DNase genes (sbcB, endA, eddB and recJ) were highly up-regulated upon L-Met treatment along with increased DNase activity in the culture supernatant. Since eDNA plays a major role in establishing and maintaining the PA biofilm, DNase activity is effective in disrupting the biofilm. Upon treatment with L-Met, the otherwise recalcitrant PA biofilm now shows susceptibility to ciprofloxacin. This was reflected in vivo, in the murine chronic PA lung infection model. Mice treated with L-Met responded better to antibiotic treatment, leading to enhanced survival as compared to mice treated with ciprofloxacin alone. These results clearly demonstrate that L-Met can be used along with antibiotic as an effective therapeutic against chronic PA biofilm infection.

Successful treatment of biofilm infections using shock waves combined with antibiotic therapy

Many bacteria secrete a highly hydrated framework of extracellular polymer matrix on suitable substrates and embed within the matrix to form a biofilm. Bacterial biofilms are observed on many medical devices, endocarditis, periodontitis and lung infections in cystic fibrosis patients. Bacteria in biofilm are protected from antibiotics and >1,000 times of the minimum inhibitory concentration may be required to treat biofilm infections. Here, we demonstrated that shock waves could be used to remove Salmonella, Pseudomonas and Staphylococcus biofilms in urinary catheters. The studies were extended to a Pseudomonas chronic pneumonia lung infection and Staphylococcus skin suture infection model in mice. The biofilm infections in mice, treated with shock waves became susceptible to antibiotics, unlike untreated biofilms. Mice exposed to shock waves responded to ciprofloxacin treatment, while ciprofloxacin alone was ineffective in treating the infection. These results demonstrate for the first time that, shock waves, combined with antibiotic treatment can be used to treat biofilm infection on medical devices as well as in situ infections.

Infiltration of Matrix-Non-producers Weakens the Salmonella Biofilm and Impairs Its Antimicrobial Tolerance and Pathogenicity

Bacterial biofilms display a collective lifestyle, wherein the cells secrete extracellular polymeric substances (EPS) that helps in adhesion, aggregation, stability, and to protect the bacteria from antimicrobials. We asked whether the BPS could act as a public good for the biofilm and observed that infiltration of cells that do not produce matrix components weakened the biofilm of Salmonella enterica serovar Typhimurium. PS production was costly for the producing cells, as indicated by a significant reduction in the fitness of wild type (WT) cells during competitive planktonic growth relative to the non-producers. Infiltration frequency of non-producers in the biofilm showed a concomitant decrease in overall productivity. It was apparent in the confocal images that the non producing cells benefit from the BPS produced by the Wild Type (WT) to stay in the biofilm. The biofilm containing non-producing cells were more significantly susceptible to sodium hypochlorite and ciprofloxacin treatment than the WT biofilm. Biofilm infiltrated with non-producers delayed the pathogenesis, as tested in a murine model. The cell types were spatially assorted, with non producers being edged out in the biofilm. However, cellulose was found to act as a barrier to keep the non-producers away from the WT microcolony. Our results show that the infiltration of non-cooperating cell types can substantially weaken the biofilm making it vulnerable to antibacterials and delay their pathogenesis. Cellulose, a component of BPS, was shown to play a pivotal role of acting as the main public good, and to edge-out the non-producers away from the cooperating microcolony.

Biomethanation of herbaceous biomass residues using 3-zone plug flow like digesters - A case study from India

Biomethanation of herbaceous biomass feedstock has the potential to provide clean energy source for cooking and other activities in areas where such biomass availability predominates. A biomethanation concept that involves fermentation of biomass residues in three steps, occurring in three zones of the fermentor is described. This approach while attempting take advantage of multistage reactors simplifies the reactor operation and obviates the need for a high degree of process control or complex reactor design. Typical herbaceous biomass decompose with a rapid VFA flux initially (with a tendency to float) followed by a slower decomposition showing balanced process of VFA generation and its utilization by methanogens that colonize biomass slowly. The tendency to float at the initial stages is suppressed by allowing previous days feed to hold it below digester liquid which permits VFA to disperse into the digester liquid without causing process inhibition. This approach has been used to build and operate simple biomass digesters to provide cooking gas in rural areas with weed and agro-residues. With appropriate modifications, the same concept has been used for digesting municipal solid wastes in small towns where large fermentors are not viable. With further modifications this concept has been used for solid-liquid feed fermentors. Methanogen colonized leaf biomass has been used as biofilm support to treat coffee processing wastewater as well as crop litter alternately in a year. During summer it functions as a biomass based biogas plants operating in the three-zone mode while in winter, feeding biomass is suspended and high strength coffee processing wastewater is let into the fermentor achieving over 90% BOD reduction. The early field experience of these fermentors is presented.

Modeling of heat and mass transfer for solid state fermentation process in tray bioreactor

A mathematical model is developed to simulate oxygen consumption, heat generation and cell growth in solid state fermentation (SSF). The fungal growth on the solid substrate particles results in the increase of the cell film thickness around the particles. The model incorporates this increase in the biofilm size which leads to decrease in the porosity of the substrate bed and diffusivity of oxygen in the bed. The model also takes into account the effect of steric hindrance limitations in SSF. The growth of cells around single particle and resulting expansion of biofilm around the particle is analyzed for simplified zero and first order oxygen consumption kinetics. Under conditions of zero order kinetics, the model predicts upper limit on cell density. The model simulations for packed bed of solid particles in tray bioreactor show distinct limitations on growth due to simultaneous heat and mass transport phenomena accompanying solid state fermentation process. The extent of limitation due to heat and/or mass transport phenomena is analyzed during different stages of fermentation. It is expected that the model will lead to better understanding of the transport processes in SSF, and therefore, will assist in optimal design of bioreactors for SSF.

Synthetic arabinomannan glycolipids and their effects on growth and motility of the Mycobacterium smegmatis

Arabinomannan-containing glycolipids, relevant to the mycobacterial cell-wall component lipoarabinomannan, were synthesized by chemical methods. The glycolipids were presented with tri- and tetrasaccharide arabinomannans as the sugar portion and a double alkyl chain as the lyophilic portion. Following synthesis, systematic biological and biophysical studies were undertaken in order to identify the effects of the glycolipids during mycobacterium growth. The studies included mycobacterial growth, biofilm formation and motility assays. From the studies, it was observed that the synthetic glycolipid with higher arabinan residues inhibited the mycobacterial growth, lessened the biofilm formation and impaired the motility of mycobacteria. A surface plasmon resonance study involving the immobilized glycan surface and the mycobacterial crude lysates as analytes showed specificities of the interactions. Further, it was found that cell lysates from motile bacteria bound oligosaccharide with higher affinity than non-motile bacteria.