Desorption of adherent bacteria from a solid hydrophobic surface by oil

Oils efficiently desorbed bacteria from a solid surface (polystyrene). Desorption was measured qualitatively by staining techniques, and quantitatively by following the outgrowth kinetics of cells remaining on the plastic surface following the washing procedure. Approximately 97% of Acinetobacter calcoaceticus RAG-1 cells adhering to a polystyrene cuvette were removed following two successive washings in the presence of olive oil.s Results suggest that oils may serve as potent, non-toxic desorbing agents to remove attached hydrophobic bacteria from surfaces.     

Growth of Thiobacillus ferrooxidans on Elemental Sulfur

Growth kinetics of Thiobacillus ferrooxidans in batch cultures, containing prills of elementary sulfur as the sole energy source, were studied by measuring the incorporation of radioactive phosphorus in free and adsorbed bacteria. The data obtained indicate an initial exponential growth of the attached bacteria until saturation of the susceptible surface was reached, followed by a linear release of free bacteria due to successive replication of a constant number of adsorbed bacteria. These adsorbed bacteria could continue replication provided the colonized prills were transferred to fresh medium each time the stationary phase was reached. The bacteria released from the prills were unable to multiply, and in the medium employed they lost viability with a half-life of 3.5 days. The spreading of the progeny on the surface was followed by staining the bacteria on the prills with crystal violet; this spreading was not uniform but seemed to proceed through distortions present in the surface. The specific growth rate of T. ferrooxidans ATCC 19859 was about 0.5 day⁻¹, both before and after saturation of the sulfur surface. The growth of adsorbed and free bacteria in medium containing both ferrous iron and elementary sulfur indicated that T. ferrooxidans can simultaneously utilize both energy sources.     

Immunolocalization of Hsp60 in Legionella pneumophila

One of the most abundant proteins synthesized by Legionella pneumophila, particularly during growth in a variety of eukaryotic host cells, is Hsp60, a member of the GroEL family of molecular chaperones. The present study was initiated in response to a growing number of reports suggesting that for some bacteria, including L. pneumophila, Hsp60 may exist in extracytoplasmic locations. Immunolocalization techniques with Hsp60-specific monoclonal and polyclonal antibodies were used to define the subcellular location and distribution of Hsp60 in L. pneumophila grown in vitro, or in vivo inside of HeLa cells. For comparative purposes Escherichia coli, expressing recombinant L. pneumophila Hsp60, was employed. In contrast to E. coli, where Hsp60 was localized exclusively in the cytoplasm, in L. pneumophila Hsp60 was predominantly associated with the cell envelope, conforming to a distribution pattern typical of surface molecules that included the major outer membrane protein OmpS and lipopolysaccharide. Interestingly, heat-shocked L. pneumophila organisms exhibited decreased overall levels of cell-associated Hsp60 epitopes and increased relative levels of surface epitopes, suggesting that Hsp60 was released by stressed bacteria. Putative secretion of Hsp60 by L. pneumophila was further indicated by the accumulation of Hsp60 in the endosomal space, between replicating intracellular bacteria. These results are consistent with an extracytoplasmic location for Hsp60 in L. pneumophila and further suggest both the existence of a novel secretion mechanism (not present in E. coli) and a potential role in pathogenesis.     

Electron microscopy of the surfaces of bacillus spores

The surface structures of the spores of Bacillus cereus, Bacillus thuringiensis, and Brevibacillus laterosporus were studied by transmission and scanning electron microscopy. Platinum deposition and negative staining with uranyl acetate revealed appendages and exosporium in B. thuringiensis and B. cereus. The exosporium structure was visualized by negative staining and ultrathin sectioning. For staining the exosporium polysaccharide, Alcian blue was used during fixation. The results obtained show the differences in structural organization of appendages and exosporium in different strains. Canoe-shaped inclusions were revealed in all Br. laterosporus strains, while strain IGM16-92 had a fibrillar capsule as well. Electron microscopy using a dual beam scanning electron microscope Quanta 200 3D provided the information of the spore surface relief without sample treatment (fixation and dehydration). The spores of Br. laterosporus strains had folded surface, unlike the smooth surface of B. cereus and B. thuringiensis spores. The diversity of external spore structures was shown within a species, which may be used for detection of bacteria at the strain level. Optimized procedures for visualization of spore surface by different electron microscopic techniques were discussed.     

Lipopeptide surfactants: Production,recovery and pore forming capacity

Lipopeptides are microbial surface active compounds produced by a wide variety of bacteria, fungi and yeast. They are characterized by highly structural diversity and have the ability to decrease the surface and interfacial tension at the surface and interface, respectively. Surfactin, iturin and fengycin of Bacillus subtilis are among the most studied lipopeptides. This review will present the main factors encountering lipopeptides production along with the techniques developed for their extraction and purification. Moreover, we will discuss their ability to form pores and destabilize biological membrane permitting their use as antimicrobial, hemolytic and antitumor agents. These open great potential applications in biomediacal, pharmaceutic and agriculture fields.     

Comparison of Atomic Force Microscopy Interaction Forces between Bacteria and Silicon Nitride Substrata for Three Commonly Used Immobilization Methods

Atomic force microscopy (AFM) has emerged as a powerful technique for mapping the surface morphology of biological specimens, including bacterial cells. Besides creating topographic images, AFM enables us to probe both physicochemical and mechanical properties of bacterial cell surfaces on a nanometer scale. For AFM, bacterial cells need to be firmly anchored to a substratum surface in order to withstand the friction forces from the silicon nitride tip. Different strategies for the immobilization of bacteria have been described in the literature. This paper compares AFM interaction forces obtained between Klebsiella terrigena and silicon nitride for three commonly used immobilization methods, i.e., mechanical trapping of bacteria in membrane filters, physical adsorption of negatively charged bacteria to a positively charged surface, and glutaraldehyde fixation of bacteria to the tip of the microscope. We have shown that different sample preparation techniques give rise to dissimilar interaction forces. Indeed, the physical adsorption of bacterial cells on modified substrata may promote structural rearrangements in bacterial cell surface structures, while glutaraldehyde treatment was shown to induce physicochemical and mechanical changes on bacterial cell surface properties. In general, mechanical trapping of single bacterial cells in filters appears to be the most reliable method for immobilization.     

Enumeration and Localization of N2-Fixing Bacteria Associated with Roots of Spartina alterniflora Loisel

Numbers and possible locations of N₂-fixing bacteria were investigated in roots of Spartina alterniflora Loisel, which support nitrogenase activity in the undisturbed native habitat. N₂-fixing bacteria were recovered in cultures both from S. alterniflora roots and from the surrounding sediment, and they formed a greater proportion of the bacteria recovered from root homogenates than from salt-marsh sediment. N₂-fixing bacteria were recovered in high numbers from the rhizoplane of S. alterniflora after roots were treated with 1 or 5% chloramine-T for 1 h or with 1% NaOCl for 1 or 2 h. Immersing S. alterniflora roots in 5% NaOCl for 1 h was more effective in distinguishing bacteria inside the roots since this treatment nearly eliminated N₂-fixing bacteria recoverable from the rhizoplane, although high numbers of N₂-fixing bacteria were recovered from homogenates of roots treated with 5% NaOCl for 1 h. However, this treatment was less effective with roots of Zea mays L. (Funks G4646) and Sorghum bicolor (L.) Moench (CK-60 A), indicating that techniques to surface sterilize roots should be evaluated for different plants. Bacteria were observed by light and electron microscopy inter- and intracellularly in the cortex and in the aerenchyma of S. alterniflora roots. This study clearly shows that bacteria, including N₂ fixers, colonize the interior of roots of S. alterniflora growing in a Chesapeake Bay, Maryland, salt marsh.     

LapF and Its Regulation by Fis Affect the Cell Surface Hydrophobicity of Pseudomonas putida

The ability of bacteria to regulate cell surface hydrophobicity is important for the adaptation to different environmental conditions. The hydrophobicity of cell surface can be determined by several factors, including outer membrane and surface proteins. In this study, we report that an adhesin LapF influences cell surface hydrophobicity of Pseudomonas putida. Cells lacking LapF are less hydrophobic than wild-type cells in stationary growth phase. Moreover, the overexpression of the global regulator Fis decreases surface hydrophobicity by repressing the expression of lapF. Flow cytometry analysis revealed that bacteria producing LapF are more viable when confronted with methanol (a hydrophilic compound) but are more susceptible to 1-octanol (a hydrophobic compound). Thus, these results revealed that LapF is the hydrophobicity factor for the cell surface of P. putida.     

Identification of nitrifiers and nitrification performance in drinking water biological activated carbon (BAC) filtration

In this study laboratory scale biological activated carbon (BAC) columns were operated with water taken from a surface water reservoir in Istanbul. The aim was to evaluate the efficiency of nitrification in columns packed with two different granular activated carbon grades (open superstructure/chemically activated and closed superstructure/steam activated carbon) and to examine the probable beneficial effect of pre-ozonation. The occurrence and diversity of ammonia-oxidizing bacteria were investigated using 16S rDNA and amoA gene based molecular techniques. Nearly complete removal of NH₄⁺-N was achieved by nitrification in both carbon types. The nitrification efficiency did not change in columns fed with ozonated water. However, the type of feed (either raw or ozonated) played a more important role than the type of GAC with respect to the dominance of nitrifier species in BAC columns. In biofilters ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were most closely related to Nitrosomonas spp. and Nitrospira spp. as determined by cloning and slot-blot analysis, respectively. The fraction of the AOB population in the biomass was high as detected by real-time PCR. The amoA/16S rDNA ratio varied from 28.7% to 2.1% along the depth of filters. In spite of similar removal efficiencies, BAC columns fed with ozonated water harbored different types of AOB than columns that were receiving raw water.     

Distribution and Identification of Luminous Bacteria from the Sargasso Sea

Vibrio fischeri and Lucibacterium harveyi constituted 75 of the 83 luminous bacteria isolated from Sargasso Sea surface waters. Photobacterium leiognathi and Photobacterium phosphoreum constituted the remainder of the isolates. Luminescent bacteria were recovered at concentrations of 1 to 63 cells per 100 ml from water samples collected at depths of 160 to 320 m. Two water samples collected at the thermocline yielded larger numbers of viable, aerobic heterotrophic and luminous bacteria. Luminescent bacteria were not recovered from surface microlayer samples. The species distribution of the luminous bacteria reflected previously recognized growth patterns; i.e., L. harveyi and V. fischeri were predominant in the upper, warm waters (only one isolate of P. phosphoreum was obtained from surface tropical waters).     

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.     

The production of tetrodotoxin-like substances by nemertean worms in conjunction with bacteria

Evidence is presented which strongly indicates a relationship between the presence of Vibrio bacteria, probably Vibrio alginolyticus, and the synthesis of tetrodotoxin (TTX)-like chemicals in seven species of British nemerteans. The occurrence of these substances and associated Vibrio bacteria in these species was investigated by bacteriological, chromatographic, spectroscopic and ultraviolet spectrometric techniques. It is suggested that these toxins are utilised by the nemerteans as a chemical defence against potential predators.     

Fast-Growing, Aerobic, Heterotrophic Bacteria from the Rhizosphere of Young Sugar Beet Plants

Fast-growing, aerobic, heterotrophic bacteria from the root surface of young sugar beet plants were inventoried. Isolation of the most abundant bacteria from the root surface of each of 1,100 plants between the second and tenth leaf stage yielded 5,600 isolates. These plants originated from different fields in Belgium and Spain. All isolates were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cellular proteins. Comparison of protein fingerprints allowed us to inventory the bacteria of individual plants of different fields or leaf stages and to analyze the composition and variability of the rhizobacterial population of young sugar beet plants. Each field harbored a specific population of bacteria which showed a highly hierarchic structure. A small number of bacteria occurring frequently at high densities dominated in each field. The major bacteria were identified as Pseudomonas fluorescens, Xanthomonas maltophilia, Pseudomonas paucimobilis, and Phyllobacterium sp. The former three species showed a high genetic variability as they were represented by different protein fingerprint types on the same or different fields or leaf stages. Twinspan analysis and relative abundance plots showed that the structure and composition of the bacterial populations varied strongly over time. Pseudomonads were typically early colonizers which were later replaced by X. maltophilia or Phyllobacterium sp.     

Quantitative Detection of Corynebacterium casei in Cheese by Real-Time PCR

The flora on the surface of smear-ripened cheeses is composed of numerous species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. Due to the absence of selective media, it is very difficult to quantify cheese surface bacteria, and, consequently, the ecology of the cheese surface microflora has not been extensively investigated. We developed a SYBR green I real-time PCR method to quantify Corynebacterium casei, a major species of smear-ripened cheeses, using primers designed to target the 16S rRNA gene. It was possible to recover C. casei genomic DNA from the cheese matrix with nearly the same yield that C. casei genomic DNA is recovered from cells recovered by centrifugation from liquid cultures. Quantification was linear over a range from 10⁵ to 10¹⁰ CFU per g of cheese. The specificity of the assay was demonstrated with DNA from species related to C. casei and from other bacteria and yeasts belonging to the cheese flora. Nine commercial cheeses were analyzed by real-time PCR, and six of them were found to contain more than 10⁵ CFU equivalents of C. casei per g. In two of them, the proportion of C. casei in the total bacterial flora was nearly 40%. The presence of C. casei in these samples was further confirmed by single-strand conformation polymorphism analysis and by a combined approach consisting of plate counting and 16S rRNA gene sequencing. We concluded that SYBR green I real-time PCR may be used as a reliable species-specific method for quantification of bacteria from the surface of cheeses.     

Efficacy of some commerical disinfectants against the bacterial isolates from a poultry slaughterhouse in Turkey

In this study, efficacy of seven different commercial disinfectant preparations was investigated against characteristic bacteria of a poultry slaughterhouse in Ankara (Turkey) by using paper disc-agar diffusion method and surface effectiveness test. According to paper disc-agar diffusion method, some disinfectants had wide efficacy against the test bacteria. The disinfectant effectiveness generally increased by the increasing of disinfectant concentration. However, some disinfectants were ineffective even though at their highest concentrations. The most effective disinfectant was B which contains QAC as active agent.Staphylococcus spp.,Staphyloccus aureus andEscherichia coli were the most sensitive bacteria to the disinfectants. Some pathogenic isolates, especiallySalmonella spp. andCampylobacter spp., were the most resistant ones to many of the disinfectants tested. The results of paper disc-agar diffusion method indicated the importance of characteristic bacterial strains of food plants as the test bacteria for disinfectant efficacy tests. Conversely of paper disc-agar diffusion method, all disinfectants were effective against the isolates by surface effectiveness test depending on exposure time. The disinfectants, except A and F, produced at least 3 log unit reduction during 5 min of exposure. However, all disinfectants at their lowest concentrations were effective against all tested bacteria during 15 and 30 min by this test.     

Optimisation of support medium for particle-based biofilm reactors

The aim of this study was to propose a method to improve the biofilm growth on different polymer materials by modifying their surface properties. The ability of two aerobic bacteria strains: Pseudomonas aeruginosa O1 and Bacillus subtilis CIP 5265 to grow on various non-coated and coated polymer materials were investigated. A layer of polymethylmethacrylate and powdered activated carbon (PMMA/PAC) was used to improve the microbial adhesion dynamics. The substratum and cell surface properties were characterized using contact angle measurements. Fluorescent microscopy and SEM were used to observe the support and the biofilm growth. It was determined that better results can be obtained increasing the difference between the surface free energies of the support and the bacteria. It was found that supports with modified surface show higher biofilm development rate and better surface colonization. The influence of the surface free energy on the detachment force and correspondingly on the biofilm formation was demonstrated.     

In Situ Analysis of Nitrifying Biofilms as Determined by In Situ Hybridization and the Use of Microelectrodes

We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in domestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these techniques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In situ hybridization revealed that bacteria belonging to the genus Nitrosomonas were the numerically dominant ammonia-oxidizing bacteria in both types of biofilms. Bacteria belonging to the genus Nitrobacter were not detected; instead, Nitrospira-like bacteria were the main nitrite-oxidizing bacteria in both types of biofilms. Nitrospira-like cells formed irregularly shaped aggregates consisting of small microcolonies, which clustered around the clusters of ammonia oxidizers. Whereas most of the ammonia-oxidizing bacteria were present throughout the biofilms, the nitrite-oxidizing bacteria were restricted to the active nitrite-oxidizing zones, which were in the inner parts of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of nitrite-oxidizing bacteria, as determined by FISH.     

Oriented Adhesion of Escherichia coli to Polystyrene Particles

The adhesion of nonflagellated Escherichia coli strain K-12 to polystyrene (PS) latex spheres or glass capillaries has been observed by using several techniques. Attention was focused on the orientation of the rod-shaped bacteria as they adhered to the surfaces in 100 mM phosphate-buffered saline. Data show that PS particles adhered to the ends of the bacteria more than 90% of the time. Moreover, the PS particles adhered to one end only, never to both. Similarly, for experiments with bacteria adhering to glass, the bacteria adhered on their ends. In order to determine whether the end of a bacterium had a different charge density from that of the middle, rotational electrophoresis experiments were used. These experiments indicated no measurable charge nonuniformity. In order to examine how strongly adhered the bacteria were to the PS particles, differential electrophoresis was used. Almost always, bacteria were found to be irreversibly adhered to the PS spheres. The cause of the oriented adhesion is not likely due to surface lipopolysaccharides (LPS), since the three strains of K-12 that were used, each having a different length of LPS, showed similar behavior. The results are discussed in terms of bacterial cell polarity. The data indicate that nanodomains on the bacterial ends are important for adhesion and that the time scale for irreversible adhesion is short.     

Node position influences viability and contamination in hazelnut shoot explants

Initiation of shoot cultures is difficult in many woody plants due to internal microbial contaminants and general lack of juvenility in material from the source plants. Hazelnuts (Corylus avellana L.) are generally difficult to initiate into culture for these same reasons. This study was designed to determine the effects of collection and surface disinfestation techniques and nodal position on the viability and contamination of shoot explants. In addition, culturable bacteria were identified on samples from surface-disinfested explants. Explants were collected from scion wood grafted onto seedling rootstocks and grown in a greenhouse. Single-node explants, excluding the shoot tip, were collected and the node location documented. After surface disinfestation, explants were held in liquid contaminant detection medium for 1 wk and the effect of this treatment on explant viability was evaluated. Node position was important for obtaining viable contaminant-free explants. Bacterial and fungal contaminations both increased with the distance from the shoot tip. The use of contaminant detection medium as a part of the initiation procedure did not affect viability. Explant-derived bacteria were identified as belonging to Brevundimonas sp. and Pseudomonas sp. through 16S rRNA sequence and API® tests. The best procedure for collecting axenic, viable hazelnut explants was to collect from the first three apical nodes, excluding the shoot tip, of actively growing greenhouse plants, place them in individual tubes for washing and surface disinfestation, and use contaminant detection techniques to identify contaminant-free cultures at initiation.     

Heterotrophic bacteria associated with the green alga <Emphasis Type="Italic">Ulva rigida</Emphasis>: identification and antimicrobial potential

Heterotrophic bacteria associated with the green alga Ulva rigida, collected from the coast of Tunisia, were isolated and subsequently identified by their 16S rRNA gene sequences and by phylogenetic analysis. The 71 isolates belong to four phyla: Proteobacteria (Alpha-and Gamma- subclasses), Bacteroidetes, Firmicutes, and Actinobacteria. Most of the isolates belong to Proteobacteria. The Gram-positive Firmicutes and especially the genus Bacillus were well-represented at the surface of U. rigida, collected from the coast as well as from the lagoon, while Actinobacteria were represented only at the surface of algae collected from the coast of Cap Zebib. Bacteroidetes were more represented at the surface of algae collected from the Ghar El Melh lagoon. The bacterial community of the water surrounding the algae was different from that associated with the surface of the algae. Moreover, the abundance of bacteria in the surrounding water was much lower compared to the density of bacteria associated with the surface of the algae. Bacteria isolated from the algal surface were tested for their antimicrobial potential. The results show that ~?36% of the algae-associated bacterial isolates possess antibacterial activity whereas free-living bacteria, isolated from the surrounding water, did not show such activity. The surface of U. rigida was colonized by a high diversity of culturable and possibly novel epiphytic bacteria that may be an important source of antimicrobial compounds and are therefore of biotechnological interest.