Bacterial deposition in porous medium as impacted by solution chemistry

Bacterial transport in porous medium was investigated by means of column experiments using typical rod-shaped bacteria of Escherichia coli and Pseudomonas fluorescens. Mobility of E. coli and P. fluorescens in silica gel decreased with increasing ionic strength of the solution. In the presence of nonionic surfactants, the mobility of E. coli and P. fluorescens increased, and this was more pronounced at lower than at higher ionic strength. Bacterial transport in the porous medium was described by the equilibrium-kinetic two-region model and bacterial deposition was assumed to occur in the kinetic adsorption region only. Quantified bacterial deposition from bacterial column breakthrough curves was related to electrostatic and Lifshitz-van der Waals interactions between bacterial cells and medium surfaces. It was found that electrostatic interactions played a more important role than Lifshitz-van der Waals interactions in determining bacterial deposition in the porous medium, and were actually the barrier for bacteria to attach to the porous medium.     

Impact of lux gene insertion on bacterial surface properties and transport

Genetic markers have been in popular use for tracing microbial movement in the environment. However, the impact of genetic marker insertion on microbial surface properties and consequent transport is often ignored. For this research, we investigated the impact of luminescence-based genetic marker insertion on bacterial surface properties and transport. Typical Gram-positive bacterial strains of Lactobacillus casei, Streptococcus mitis and Micrococcus luteus were used as model bacterial strains in this research. We manipulated gene transfer to observe the impact of lux gene insertion on bacterial surface properties based on contact angle measurements, and we conducted column experiments to evaluate the impact of lux gene insertion on bacterial transport. After lux gene insertion, bacterial interactions with the porous media increased, demonstrating stronger deposition potential in the porous media. Accordingly, retention of the daughter strains increased. Lux gene insertion also resulted in an increase in bacterial dispersion and equilibrium adsorption in the porous media. The bacterial deposition coefficient was found to correlate with the free energy of interactions between bacteria and the porous media.     

Rhamnolipid biosurfactant behavior in solutions

The solution behavior of rhamnolipid biosurfactant that is determined by its surface thermodynamic properties often plays a central role in determining and controlling its performance in practical applications. In this study, rhamnolipid molecules exhibited a predominant hydrophilic surface, contributed by hydrophilic monomer head groups. Interaction free energies between rhamnolipid molecules at water surfaces, as estimated based on the distance-dependent decaying interaction contact areas, decayed exponentially with regards to separation distance. These interaction free energies were governed by hydrophilic monomer head groups of rhamnolipid molecules. The experimental observations in this study were consistent with the previous postulations.     

Microbial diversity of culturable heterotrophs in the rhizosphere of salt marsh grass, Porteresia coarctata (Tateoka) in a mangrove ecosystem

A study was conducted to understand the complexity of bacterial diversity of rhizosphere of Porteresia coarctata based on culture dependent method. A large number of bacteria were isolated on nutrient agar medium supplemented with 1% NaCl and the dominant ones were further analyzed with PCR-RFLP method. The sequence analyses of the dominant strains revealed that most of the sequences belonged to members of gamma proteobacteria, firmicutes, bacteroidetes and uncultured bacteria. The phylogenetic analysis of 16S rRNA gene sequences revealed close relationships to a wide range of clones or bacterial species of various divisions. These results afford an understanding of the role of rhizobacteria in alleviating salt stress in Porteresia coarctata expected to contribute towards long-term goal of improving plant-microbe interactions for salinity affected fields.     

Effect of biosurfactant derived from Vibrio natriegens MK3 against Vibrio harveyi biofilm and virulence

Vibrio harveyi is a marine luminous pathogen, which causes biofilm‐mediated infections, pressures the search for an innovative alternate approach to strive against vibriosis in aquaculture. This study anticipated to explore the effect of glycolipid biosurfactant as an antipathogenic against V. harveyi to control vibriosis. In this study, 27 bacterial strains were isolated from marine soil sediments. Out of these, 11 strains exhibited surfactant activity and the strain MK3 showed high emulsification index. The potent strain was identified as Vibrio natriegens and named as V. natriegens MK3. The extracted biosurfactant was purified using high‐performance liquid chromatography and it was efficient to decrease the surface tension of the growth medium up to 21 mN/m. The functional group and composition of the biosurfactant were determined by Fourier‐transform infrared spectroscopy and nuclear magnetic resonance spectroscopy spectral studies and the nature of the biosurfactant was identified as glycolipid. The surfactant was capable of reducing the biofilm formation, bioluminescence, extracellular polysaccharide synthesis, and quorum sensing in marine shrimp pathogen V. harveyi. The antagonistic effect of biosurfactant was evaluated against V. harveyi‐infected brine shrimp Artemia salina. This study reveals that biosurfactant can be considered for the management of biofilm‐related aquatic infections.     

Gentamicin-eluting bioresorbable composite fibers for wound healing applications

New gentamicin-eluting bioresorbable core/shell fiber structures were developed and studied. These structures were composed of a polyglyconate core and a porous poly(DL-lactic-co-glycolic acid) (PDLGA) shell loaded with the antibiotic agent gentamicin, prepared using freeze drying of inverted emulsions. These unique fibers are designed to be used as basic elements of bioresorbable burn and ulcer dressings. The investigation focused on the effects of the emulsion's composition (formulation) on the shell's microstructure, on the drug release profile from the fibers, and on bacterial inhibition. The release profiles generally exhibited an initial burst effect accompanied by a decrease in release rates with time. Albumin was found to be the most effective surfactant for stabilizing the inverted emulsions. All three formulation parameters had a significant effect on gentamicin's release profile. An increase in the polymer and organic:aqueous phase ratio or a decrease in the drug content resulted in a lower burst release and a more moderate release profile. The released gentamicin also resulted in a significant decrease in bacterial viability and practically no bacteria survived after 2 days when using bacterial concentrations of 1 x 10(7) CFU/mL. Thus, our new fiber structures are effective against the relevant bacterial strains and can be used as basic elements of bioresorbable drug-eluting wound dressings.     

Contribution of Choline-Binding Proteins to Cell Surface Properties of Streptococcus pneumoniae

Nonspecific interactions related to physicochemical properties of bacterial cell surfaces, such as hydrophobicity and electrostatic charge, are known to have important roles in bacterium-host cell encounters. Streptococcus pneumoniae (pneumococcus) expresses multiple, surface-exposed, choline-binding proteins (CBPs) which have been associated with adhesion and virulence. The purpose of this study was to determine the contribution of CBPs to the surface characteristics of pneumococci and, consequently, to learn how CBPs may affect nonspecific interactions with host cells. Pneumococcal strains lacking CBPs were derived by adapting bacteria to a defined medium that substituted ethanolamine for choline. Such strains do not anchor CBPs to their surface. Cell surface hydrophobicity was tested for the wild-type and adapted strains by using a biphasic hydrocarbon adherence assay, and electrostatic charge was determined by zeta potential measurement. Adherence of pneumococci to human-derived cells was assessed by fluorescence-activated cell sorter analysis. Strains lacking both capsule and CBPs were significantly more hydrophobic than nonencapsulated strains with a normal complement of CBPs. The effect of CBPs on hydrophobicity was attenuated in the presence of capsule. Removal of CBPs conferred a greater electronegative net surface charge than that which cells with CBPs possessed, regardless of the presence of capsule. Strains that lack CBPs were poorly adherent to human monocyte-like cells when compared with wild-type bacteria with a full complement of CBPs. These results suggest that CBPs contribute significantly to the hydrophobic and electrostatic surface characteristics of pneumococci and may facilitate adherence to host cells partially through nonspecific, physicochemical interactions.     

Molecular and phenotypic characterization of Acinetobacter strains able to degrade diesel fuel

Characterization of bacterial communities in oil-contaminated soils and evaluation of their degradation capacities may serve as a guide for improving remediation of such environments. Using physiological and molecular methods, the aim of this work was to characterize 17 Acinetobacter strains (13 species) able to use diesel fuel oil as sole carbon and energy source. The strains were first tested for their ability to grow on different alkanes on minimal medium containing high NaCl concentrations. The envelope hydrophobicity of each strain was assessed by microbial adhesion to the hydrocarbon test (MATH) when grown in LB medium or minimal medium containing succinate or diesel fuel. Most strains were hydrophobic both in LB and minimal medium, except for strain Acinetobacter venetianus VE-C3 that was hydrophobic only in minimal medium. Furthermore, two A. venetianus strains, RAG-1(T) and LUH 7437, and strain ATCC 17905 (genomic species 13BJ) displayed biosurfactant activity. The alkM gene encoding alkane hydroxylase was detected in the chromosome of the 15 strains by PCR amplification, sequencing and Southern blot analysis. Phenotype microarray analysis performed on the five A. venetianus strains revealed that they differentially used purines as N-source and confirmed that they are unable to use carbohydrates.     

In vitro colonization of Streptococcus mutans on enamel.

An in vitro model consisting of enamel from extracted human molars, suspended from wires in inoculated culture tubes, was used to study the adhesion of bacteria to enamel. Under conditions in which there was no macroscopically visible plaque formation, electron micrographs showed no bacterial deposits on the enamel surface. In samples where Streptococcus mutans attached to enamel, an extracellular, pellicle-like material was associated with the bacteria adjacent to the enamel. This material appeared to bind to the enamel surface and to mediate bacterial attachment. Membrane-filtered (Millipore Corp.) saliva deposited a thin surface layer on the enamel, but there were no observable alterations of S. mutans attachment to enamel pretreated with saliva. It was noted that Bratthall serotype c and e strains of S. mutans, when grown in glucose-containing medium, attached, although less tenaciously, to enamel and nichrome wires. Chemical and gas chromatographic analyses of cell-associated materials formed by serotype c and e strains cultured in glucose-containing medium revealed low amounts of glucose-positive material and no polymer linkages characteristic of glucan; yet the same strains cultured in sucrose-containing medium had relatively high amounts of glucose-positive material, with polymer linkages-characteristic of glucan. Serotype a, b, and d strains could attach only in sucrose-containing media.     

Characterization and identification of an iron-oxidizing,Leptospirillum-like bacterium,present in the high sulfate leaching solution of a commercial bioleaching plant

Most copper bioleaching plants operate with a high concentration of sulfate salts, caused by the continuous addition of sulfuric acid and the recycling of the leaching solution. Since the bacteria involved in bioleaching have been generally isolated at low sulfate concentrations, the bacterial population present in the high-sulfate (150 gl(-1)) leaching solution, employed in a copper production plant, was investigated. The iron-oxidizing bacteria able to grow in the leaching solution were enriched by several batch cultivations and, after serial dilution, an abundant bacterial strain was isolated. This strain, called LA, exhibited a relatively constant rate of iron-oxidation in media containing sulfate ions at concentrations ranging from 10 to 150 gl(-1). Culture collection strains of Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans showed limited abilities to grow at sulfate ion concentrations higher than 70 gl(-1). In spite of its tolerance to high sulfate concentrations, strain LA was as sensitive to NaCl as A. ferrooxidans. Comparative sequence analysis of the 16S rRNA gene of strain LA indicated that it is phylogenetically related to strains described as Leptospirillum ferrooxidans. Bacterial community DNA restriction patterns of 16S rRNA genes suggested that strain LA was a minor component of the bacterial population present in leaching solution, but is abundant in ore leached with this solution.     

Solubility and bioactivity of the Pseudomonas quinolone signal are increased by a Pseudomonas aeruginosa-produced surfactant

Pseudomonas aeruginosa is a gram-negative bacterium that causes serious infections in immunocompromised individuals and cystic fibrosis patients. This opportunistic pathogen controls many of its virulence factors and cellular functions through the activity of three cell-to-cell signals, N-(3-oxododecanoyl)-L-homoserine lactone, N-butyryl-L-homoserine lactone, and the Pseudomonas quinolone signal (PQS). The activity of these signals is dependent upon their ability to dissolve in and freely diffuse through the aqueous solution in which P. aeruginosa happens to reside. Despite this, our data indicated that PQS was relatively insoluble in aqueous solutions, which led us to postulate that P. aeruginosa could be producing a PQS-solubilizing factor. In this report, we show that the P. aeruginosa-produced biosurfactant rhamnolipid greatly enhances the solubility of PQS in aqueous solutions. The enhanced solubility of PQS led to an increase in PQS bioactivity, as measured by both a gene induction assay and an apoptosis assay. This is the first demonstration of the importance of a bacterial surfactant in the solubilization and bioactivity of a cell-to-cell signal.     

Antibiotics enhance binding by human lipid A-reactive monoclonal antibody HA-1A to smooth gram-negative bacteria.

The effect of antibiotic exposure of phenotypically smooth gram-negative bacteria on binding by the human lipid A-reactive monoclonal antibody HA-1A (trademark of Centocor, Inc.) was examined by liquid-phase immunoassay and by dual-parameter flow cytometry (fluorescence-activated cell sorter [FACS]) analysis. HA-1A exhibited dose-dependent binding to untreated rough gram-negative bacteria such as the Escherichia coli D21F2 Re chemotype strain but little binding to untreated smooth strains such as E. coli O111:B4, or to gram-positive bacteria. However, overnight incubation of E. coli O111:B4 with inhibitory concentrations of ceftazidime produced dose-dependent enhancement of HA-1A binding. Similar augmentation of HA-1A binding was observed when other smooth strains were exposed to cell wall-active agents. Dual-parameter FACS analysis of E. coli O111:B4 exposed overnight to two times the MIC of ceftazidime revealed a decrease in forward light scatter, indicating a reduction in average cell size or bacterial fragmentation, accompanied by a striking increase in lipid A-inhibitable HA-1A binding. Moreover, ceftriaxone, but not gentamicin, produced a marked increase in propidium iodide uptake, indicating an increase in bacterial cell permeability, and a corresponding enhancement of HA-1A binding. Antibiotic-induced enhancement of HA-1A binding to smooth strains of gram-negative bacteria thus appears related to specific alterations in bacterial cell morphology resulting in exposure of the epitope recognized by HA-1A.     

Interactions of Neisseria meningitidis with human monocytes

The roles of capsule, pill and Class 5 outer-membrane proteins (Opa and Opc) of Neisseria meningitidis (Nm) in bacterial interactions with human monocytes were investigated using several meningococcal isolates of different serogroups. The presence of either Class I or Class II pili in capsulate strains of several serogroups had no significant effect on adherence to and internalisation by monocytes. Using clonal variants derived from a non-piliated serogroup A strain, C751, it was observed that capsulate bacteria (cap⁺) failed to interact with human monocytes in significant numbers whether or not they expressed outer-membrane proteins. These bacteria were also resistant to phagocytic killing. For capsule-deficient bacteria, expression of the Opc protein or OpaB_c751 correlated with high levels of association, while the expression of OpaD_c751 or OpaA_c751 resulted in comparatively lower levels. Bacteria expressing undetectable levels of Opc or Opa proteins (Opc^-, Opa) failed to interact with monocytes. In phagocytic killing assays, Opc-expressing bacteria (Opc⁺) as well as Opa-expressing bacteria (Opa⁺) were killed more readily than Opc^-, Opa bacteria (30% decrease in viability of Opc⁺ bacteria; 18%, 10% and 8% decrease in viability of OpaB⁺, OpaD⁺ and OpaA⁺ bacteria). A study of intracellular survival showed a gradual decrease in viability of both capsulate and capsule-deficient bacteria. However, proportionately greater numbers of capsule-deficient bacteria were internalized and consequently larger numbers survived over a 4-h period. Prolonged bacterial survival within phagocytic cells may have implications in dissemination of bacteria by carriage within these cells.     

Bacteria related to the nitrogen cycle in salt-affected soils of Argentina

We studied abundance as well as some morphological and physiological characteristics of six groups of bacteria (total aerobic, proteolytics, ammonifiers, nitrifiers, denitrifiers and bacteria from N-free medium) in six toposequences with salt-affected soils in the semiarid region of Argentina. Abundance of most groups of bacteria was positively related with soil organic matter content and inversely related with soil pH (within a pH range of 7 to 10.5), exchangeable sodium and soil depth. CaCO₃ and electric conductivity were only related to the abundance of some bacterial groups in A and C horizons. Predictive equations for each toposequence explained between 31 and 89% of the variance in bacterial abundance. Multivariate relationships between bacterial groups and soil factors also indicated that organic matter content and pH were more important factors to influence microbial abundance than salt content in these soils. All the groups examined were present in the salt crusts which reached electric conductivity values of up to 216 dS/m. Bacillus spp. and coryneform bacteria prevailed among the 537 strains isolated from A to C horizons. Neither Azotobacter nor halophilic bacteria were found with the culture media utilized. One third of the 42 strains which grew in N-free medium utilized D -glucose and 55% did not use the sugars tested. All bacterial groups were more affected by NaCl than by Na₂SO₄ in laboratory tests with no groups surviving NaCl concentrations of 100 g/l.     

Detection of protein A-like substances on haemolytic streptococci prior to use in mixed reverse passive antiglobulin haemagglutination (MRPAH).

Before mixed reverse passive antiglobulin haemagglutination tests (MRPAH) can be used to measure the class of bacterial antibodies, the bacteria have to be shown to be free of Protein A or Protein A-like substances on their surfaces. Two basic procedures have been examined: haemagglutination of red cells coated with immunoglobulin by the bacteria, and the MRPAH reaction itself to reveal absorption of purified gamma Fc by the bacterial suspension. The use of a purified gamma Fc component has proved successful in providing a sensitive test for the detection of Protein A-like substances on the surface of bacterial. In addition to both the Cowan and Wood strains of Staph, aureus, strains of haemolytic streptococci of groups A, C and G had Protein A-like substances on their surfaces. In contrast, strains of group B and group D, as well as Strep. milleri, had no detectable Protein A-like activity.     

Adherence of Pseudomonas aeruginosa to human tracheobronchial mucin.

A microtiter plate assay was developed to study the adherence of Pseudomonas aeruginosa to purified human tracheobronchial mucin. The wells of the plates were treated with silicon to minimize nonspecific binding of bacteria and then coated with a solution of purified human tracheobronchial mucin. Bacteria were added to the wells, and the plates were incubated at 37 degrees C. The wells were washed 15 times in an automated microtiter plate washer, and the bacteria bound to wells were desorbed with Triton X-100 and plated for enumeration. Scanning electron microscopy verified bacterial adherence to the mucin-coated wells and desorption of bacteria by Triton X-100. Adherence of P. aeruginosa increased as the concentration of mucin used to coat the wells was increased, with saturation occurring at 0.5 microgram of mucin protein per ml. Other parameters that affected adherence included the time of incubation and concentration of bacteria. Similar studies with strains of Escherichia coli and Klebsiella pneumoniae indicated a relative lack of binding of these bacteria to mucin. In comparing different strains of P. aeruginosa, there were small differences in binding between strains. It is inferred that there may be specific sites on human tracheobronchial mucin which facilitate this preferential binding.     

Vibrio cholerae-induced cellular responses of polarized T84 intestinal epithelial cells are dependent on production of cholera toxin and the RTX toxin.

To study the utility of in vitro-polarized intestinal cell monolayers for modeling Vibrio cholerae-host cell interactions, we added live V. cholerae bacteria to the apical surfaces of polarized T84 cell monolayers and monitored changes in electrical properties. We found that both classical and El Tor strains produce cholera toxin after addition to the monolayer, but induction is most likely due to medium components rather than bacterium-cell interactions. We also found that the RTX toxin is produced by El Tor strains. This toxin caused a loss of the barrier function of the paracellular tight junction that was measured as a decrease in transepithelial resistance. This decrease occurred when bacteria were added to either the apical or basolateral surfaces, indicating that the RTX toxin receptor is expressed on both surfaces. These results are discussed with regard to the applicability of the polarized T84 cell monolayers as an in vitro model of host-pathogen interactions.     

The phagosomal environment protects virulent Mycobacterium avium from killing and destruction by clarithromycin.

Murine bone marrow-derived macrophages (Mphis) infected with virulent strains of Mycobacterium avium (TMC 724 and a human clinical isolate) or with an avirulent opaque variant that spontaneously dissociates from the virulent human clinical isolate were subjected to a prolonged and continuous treatment with clarithromycin added at the MIC. The efficiency of this antibiotic in terms of inhibition of bacterial growth and bacterial degradation was evaluated during a 21-day treatment period. Growth was assessed by determination of CFU of intracellular bacteria and by a quantitative ultrastructural analysis which allowed us also to determine the extent of bacterial degradation. A similar treatment was applied to the same strains growing in liquid medium. Our data show that in liquid medium, clarithromycin caused a 90% decrease in CFU within 7 days of treatment. When applied to Mphis infected with virulent M. avium, clarithromycin immediately arrested bacterial growth but was unable to fully kill and degrade intracellularly growing virulent bacteria. After 21 days of treatment, 25% of intracellular bacteria were still morphologically intact. These bacteria resumed growth upon removal of the antibiotic, with a normal replication rate. These bacteria had not become more resistant to the drug, since the MIC was unchanged as compared to the one determined for the initial stock used to infect Mphis. Our data therefore suggest that the intraphagosomal environment protects bacteria from degradation. We propose that the inability of the drug to completely destroy bacteria is the result of a limited accessibility of the drug due to prevention of fusions between the immature phagosomes in which virulent bacteria reside and lysosomes in which clarithromycin accumulates. In accord with our proposal, we show that the avirulent opaque variant, which does not prevent phagosome-lysosome fusions (unpublished data), is finally destroyed by clarithromycin even within the phagosomal environment.     

Inhibition by nifurzide of the adhesion capacity of enteropathogenic colibacillus to HEP-2 cells

Enteropathogenic Escherichia coli (EPEC) adhere in vivo to enterocytes. This adhesion capacity can be obtained in vitro with Hep-2 cells on which a characteristic localized adherence (LA) is observed. We studied the effect of subinhibitory concentrations (SIC) of nifurzide, a nitrofurane derivative, on this bacterial adherence phenomenon. Three EPEC strains are used: 11201 (026 serogroup), 7958 (0128) and 7836 (0142). Various SIC (MIC/2; MIC/4;...; MIC/32) were added either to the culture medium of bacteria or to the Eagle medium in which bacteria and Hep-2 cells were mixed during the adhesion experiments. In each case an adhesion index is determined. Nifurzide strongly inhibits the adherence capacity of the three strains when concentrations ranging from MIC/2 to MIC/16 where added in both culture media. On the other hand three other nitrofurane derivatives which have no antibacterial effect did not inhibit adherence. The mechanism of the adherence inhibition by nifurzide is discussed.     

Adsorption chromatography of polioviruses on porous glass

The paper presents a new procedure for the purification and separation of viruses based on true adsorption chromatography of virions on porous glass. By means of this method a poliovirus suspension is purified 1000-fold from all components of the culture medium and is concentrated 10-fold at the same time. A separation of two poliovirus strains is also effected. The conditions of adsorption and the transition to elution are very critical and depend on 2 kinds of molecular forces: hydrogen bonds, which can be overcome by the addition of urea, and ionic interactions, which are used to effect desorption when the surface becomes negatively charged.