Effect of Different Concentrations of Ortho-phthalaldehyde on Biofilms Formed by Pseudomonas fluorescens Under Different Flow Conditions

The effectiveness of different concentrations of ortho-phthalaldehyde (OPA) in controlling biofilms of Pseudomonas fluorescens formed on stainless steel slides, using flow cell reactors under laminar and turbulent flow, was investigated by determining the variation in mass and respiratory activity. The physical stability of the biofilm with and without exposure to OPA was studied in a rotating device as variation in the mass of the biofilm on the surface after exposure to different rotation velocities. The activity of OPA against bacterial suspended cultures was evaluated in the presence and absence of bovine serum albumin (BSA) in order to evaluate the interference of proteins on the activity of the biocide. The results showed that biofilms formed under different flow conditions had different properties and reacted differently after biocide application. Biofilms formed under laminar flow were more easily inactivated than those formed under turbulent conditions. However, OPA did not promote the detachment of biofilms from the surface. The exposure of biofilms to different shear stress conditions after OPA treatment enhanced removal from the surface, indicating that OPA may weaken the biofilm matrix. The biocide was more effective on suspended cells than on cells grown in biofilms. This fact may be explained by the reaction of the biocide with proteins of the polymeric matrix of the biofilm as suggested by the significant reduction of biocide action on suspended cells in the presence of BSA.     

Planktonic Replication Is Essential for Biofilm Formation by Legionella pneumophila in a Complex Medium under Static and Dynamic Flow Conditions

Legionella pneumophila persists for a long time in aquatic habitats, where the bacteria associate with biofilms and replicate within protozoan predators. While L. pneumophila serves as a paradigm for intracellular growth within protozoa, it is less clear whether the bacteria form or replicate within biofilms in the absence of protozoa. In this study, we analyzed surface adherence of and biofilm formation by L. pneumophila in a rich medium that supported axenic replication. Biofilm formation by the virulent L. pneumophila strain JR32 and by clinical and environmental isolates was analyzed by confocal microscopy and crystal violet staining. Strain JR32 formed biofilms on glass surfaces and upright polystyrene wells, as well as on pins of “inverse” microtiter plates, indicating that biofilm formation was not simply due to sedimentation of the bacteria. Biofilm formation by an L. pneumophila fliA mutant lacking the alternative sigma factor σ²⁸ was reduced, which demonstrated that bacterial factors are required. Accumulation of biomass coincided with an increase in the optical density at 600 nm and ceased when the bacteria reached the stationary growth phase. L. pneumophila neither grew nor formed biofilms in the inverse system if the medium was exchanged twice a day. However, after addition of Acanthamoeba castellanii, the bacteria proliferated and adhered to surfaces. Sessile (surface-attached) and planktonic (free-swimming) L. pneumophila expressed β-galactosidase activity to similar extents, and therefore, the observed lack of proliferation of surface-attached bacteria was not due to impaired protein synthesis or metabolic activity. Cocultivation of green fluorescent protein (GFP)- and DsRed-labeled L. pneumophila led to randomly interspersed cells on the substratum and in aggregates, and no sizeable patches of clonally growing bacteria were observed. Our findings indicate that biofilm formation by L. pneumophila in a rich medium is due to growth of planktonic bacteria rather than to growth of sessile bacteria. In agreement with this conclusion, GFP-labeled L. pneumophila initially adhered in a continuous-flow chamber system but detached over time; the detachment correlated with the flow rate, and there was no accumulation of biomass. Under these conditions, L. pneumophila persisted in biofilms formed by Empedobacter breve or Microbacterium sp. but not in biofilms formed by Klebsiella pneumoniae or other environmental bacteria, suggesting that specific interactions between the bacteria modulate adherence.     

Systematic Analysis of Diguanylate Cyclases That Promote Biofilm Formation by Pseudomonas fluorescens Pf0-1

Cyclic di-GMP (c-di-GMP) is a broadly conserved, intracellular second-messenger molecule that regulates biofilm formation by many bacteria. The synthesis of c-di-GMP is catalyzed by diguanylate cyclases (DGCs) containing the GGDEF domain, while its degradation is achieved through the phosphodiesterase activities of EAL and HD-GYP domains. c-di-GMP controls biofilm formation by Pseudomonas fluorescens Pf0-1 by promoting the cell surface localization of a large adhesive protein, LapA. LapA localization is regulated posttranslationally by a c-di-GMP effector system consisting of LapD and LapG, which senses cytoplasmic c-di-GMP and modifies the LapA protein in the outer membrane. Despite the apparent requirement for c-di-GMP for biofilm formation by P. fluorescens Pf0-1, no DGCs from this strain have been characterized to date. In this study, we undertook a systematic mutagenesis of 30 predicted DGCs and found that mutations in just 4 cause reductions in biofilm formation by P. fluorescens Pf0-1 under the conditions tested. These DGCs were characterized genetically and biochemically to corroborate the hypothesis that they function to produce c-di-GMP in vivo. The effects of DGC gene mutations on phenotypes associated with biofilm formation were analyzed. One DGC preferentially affects LapA localization, another DGC mainly controls swimming motility, while a third DGC affects both LapA and motility. Our data support the conclusion that different c-di-GMP-regulated outputs can be specifically controlled by distinct DGCs.     

Energy dispersive x-ray analysis of the elemental composition of Legionella pneumophila,Escherichia coli,Pseudomonas aeruginosa,and the green algae Scenedesmus sp., Chlorella sp., and Gloeocystis sp.

Energy dispersive x-ray analysis of the elemental composition of algae and bacteria cultures showed evidence of increased concentration of Mg and P in some instances and not in others.     

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions¹. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions^2,3.The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.     

Development of Heterodera glycines Ichinohe on Soybean,Glycine max (L.) Merr., under Gnotobiotic Conditions

The life cycle of the soybean cyst nematode, Race 3 (SCN 3), Heterodera glycines Inchinohe was determined from observations of the developmental stages on soybean Glycine max cv. Kent root explants under gnotobiotic conditions at 25 C. Approximately 51% of the second-stage larvae penetrated the root l day after inoculation (DAI). Third-stage larvae appeared 4 DAI, became sexually differentiated 5 DAI, and protruded from the root tissues 6 DAI. Fourth-stage males and females were observed 7 DAI. Ensheathcd adult males were observed at 9 DAI and exsheathed to free adults at 11 DAI. The fourth-stage female became an adult at l0 DAI, Males entwined arotmd the gelatinons sac of the female at 12 DAI and were assumed to be mating. Some males actually penetrated and were enveloped by the gelatinous sac. The female-to-male sex ratio ranged from 2.3 to 9.5:1. First- and second-stage larvae were observed in the egg 17 and 19 DAI, respectively. The life cycle of the SCN 3 was completed 21 DAI upon hatching of the eggs and emergence of second-stage larvae. The average number of eggs in the cyst body and gelatinous sac, was 210 and 187, respectively. Key words: reproduction, soybean cyst nematode, scanning electron microscopy.     

Streptococcus oligofermentans Inhibits Streptococcus mutans in Biofilms at Both Neutral pH and Cariogenic Conditions

Homeostasis of oral microbiota can be maintained through microbial interactions. Previous studies showed that Streptococcus oligofermentans, a non-mutans streptococci frequently isolated from caries-free subjects, inhibited the cariogenic Streptococcus mutans by the production of hydrogen peroxide (HP). Since pH is a critical factor in caries formation, we aimed to study the influence of pH on the competition between S. oligofermentans and S. mutans in biofilms. To this end, S. mutans and S. oligofermentans were inoculated alone or mixed at 1:1 ratio in buffered biofilm medium in a 96-well active attachment model. The single- and dual-species biofilms were grown under either constantly neutral pH or pH-cycling conditions. The latter includes two cycles of 8 h neutral pH and 16 h pH 5.5, used to mimic cariogenic condition. The 48 h biofilms were analysed for the viable cell counts, lactate and HP production. The last two measurements were carried out after incubating the 48 h biofilms in buffers supplemented with 1% glucose (pH 7.0) for 4 h. The results showed that S. oligofermentans inhibited the growth of S. mutans in dual-species biofilms under both tested pH conditions. The lactic acid production of dual-species biofilms was significantly lower than that of single-species S. mutans biofilms. Moreover, dual-species and single-species S. oligofermentans biofilms grown under pH-cycling conditions (with a 16 h low pH period) produced a significantly higher amount of HP than those grown under constantly neutral pH. In conclusion, S. oligofermentans inhibited S. mutans in biofilms not only under neutral pH, but also under pH-cycling conditions, likely through HP production. S. oligofermentans may be a compelling probiotic candidate against caries.     

Design of a Simple Model of <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilms Formed under Conditions of Flow: Development,Architecture, and Drug Resistance

Candida albicans biofilms on most medical devices are exposed to a flow of body fluids that provide water and nutrients to the fungal cells. While C. albicans biofilms grown in vitro under static conditions have been exhaustively studied, the same is not true for biofilms developed under continuous flow of replenishing nutrients. Here, we describe a simple flow biofilm (FB) model that can be built easily with materials commonly available in most microbiological laboratories. We demonstrate that C. albicans biofilms formed using this flow system show increased architectural complexity compared to biofilms grown under static conditions. C. albicans biofilms under continuous medium flow grow rapidly, and by 8 h show characteristics similar to 24 h statically grown biofilms. Biomass measurements and microscopic observations further revealed that after 24 h of incubation, FB was more than twofold thicker than biofilms grown under static conditions. Microscopic analyses revealed that the surface of these biofilms was extremely compact and wrinkled, unlike the open hyphal layer typically seen in 24 h static biofilms. Results of antifungal drug susceptibility tests showed that C. albicans cells in FB exhibited increased resistance to most clinically used antifungal agents.     

Marinobacter salarius sp. nov. and Marinobacter similis sp. nov., Isolated from Sea Water

Two non-pigmented, motile, Gram-negative marine bacteria designated R9SW1^T and A3d10^T were isolated from sea water samples collected from Chazhma Bay, Gulf of Peter the Great, Sea of Japan, Pacific Ocean, Russia and St. Kilda Beach, Port Phillip Bay, the Tasman Sea, Pacific Ocean, respectively. Both organisms were found to grow between 4°C and 40°C, between pH 6 to 9, and are moderately halophilic, tolerating up to 20% (w/v) NaCl. Both strains were found to be able to degrade Tween 40 and 80, but only strain R9SW1^T was found to be able to degrade starch. The major fatty acids were characteristic for the genus Marinobacter including C_16:0, C_16:1 ω7c, C_18:1 ω9c and C_18:1 ω7c. The G+C content of the DNA for strains R9SW1^T and A3d10^T were determined to be 57.1 mol% and 57.6 mol%, respectively. The two new strains share 97.6% of their 16S rRNA gene sequences, with 82.3% similarity in the average nucleotide identity (ANI), 19.8% similarity in the in silico genome-to-genome distance (GGD), 68.1% similarity in the average amino acid identity (AAI) of all conserved protein-coding genes, and 31 of the Karlin''s genomic signature dissimilarity. A phylogenetic analysis showed that R9SW1^T clusters with M. algicola DG893^T sharing 99.40%, and A3d10^T clusters with M. sediminum R65^T sharing 99.53% of 16S rRNA gene sequence similarities. The results of the genomic and polyphasic taxonomic study, including genomic, genetic, phenotypic, chemotaxonomic and phylogenetic analyses based on the 16S rRNA, gyrB and rpoD gene sequence similarities, the analysis of the protein profiles generated using MALDI-TOF mass spectrometry, and DNA-DNA relatedness data, indicated that strains R9SW1^T and A3d10^T represent two novel species of the genus Marinobacter. The names Marinobacter salarius sp. nov., with the type strain R9SW1^T ( =  LMG 27497^T  =  JCM 19399^T  =  CIP 110588^T  =  KMM 7502^T) and Marinobacter similis sp. nov., with the type strain A3d10^T ( =  JCM 19398^T  =  CIP 110589^T  =  KMM 7501^T), are proposed.     

Nacobbodera chitwoodi,n. gen., n. sp., (Nacobbidae:Nematoda) on Douglas Fir in Oregon

Nacobbodera chitwoodi, n. gen., n. sp., representing Nacobboderinae n. subfam, in the Nacobbidae, is described and illustrated from roots of Douglas fir, Pseudotsuga menziesii near Florence Oregon. This new species, having characters common to Nacobbidae, Heteroderidae, and Meloidogynidae, appears to represent a connecting link between these three families of the Heteroderoidea. Especially, a distinct tail on swollen adult females, vermiform shape of juvenile females, and the shape and sclerotization of the head of the various stages, indicate a Nacobbidae relationship. Certain other characteristics present in this new species are of the Heteroderidae and Meloidogynidae type as follows: Gross shape of adult female in posterior half of body, two ovaries, anterior position of excretory pore as in Meloidogyne females, absence of a bursa, and development of male within a sausage-shaped cuticle as in root-knot and cyst nematodes.     

Synthesis of Extracellular Proteinase by Pseudomonas fluorescens Under Conditions of Limiting Carbon, Nitrogen, and Phosphate

The influence of carbon, nitrogen, and phosphate concentrations on growth and proteinase production by Pseudomonas fluorescens 32A was examined. In mineral salts medium containing dialyzed skim milk supernatant as an inducer, maximum growth was obtained at 1.0 and 2.5 mM orthophosphate at 20 and 5°C, respectively. At both temperatures, 5 mM orthophosphate was required for maximum proteinase production, whereas significant inhibition was found at 10 mM. Orthophosphate was the only phosphate compound able to support growth. With sodium pyruvate as the carbon source, maximum enzyme synthesis was at 100 mM carbon at both temperatures. At both 20 and 5°C maximum growth and enzyme production was found with 10 mM NH₄Cl. A bioassay for available phosphate based on the growth of P. fluorescens 32A in phosphate-limited mineral salts medium showed that skim milk and skim milk supernatant contained 50 and 10 mM orthophosphate, respectively. Proteinase production in skim milk was 2.6- and 12-fold greater than that in optimal mineral salts medium at 20 and 5°C, respectively. These results suggest that proteinase production in milk does not occur as a result of nutrient limitation and may be regulated in part by milk phosphates.     

Interaction between Medicago truncatula and Pseudomonas fluorescens: Evaluation of Costs and Benefits across an Elevated Atmospheric CO2

Soil microorganisms play a key role in both plants nutrition and health. Their relation with plant varies from mutualism to parasitism, according to the balance of costs and benefits for the two partners of the interaction. These interactions involved the liberation of plant organic compounds via rhizodeposition. Modification of atmospheric CO₂ concentration may affect rhizodeposition and as a consequence trophic interactions that bind plants and microorganisms. Positive effect of elevated CO₂ on plants are rather well known but consequences for micoorganisms and their interactions with plants are still poorly understood. A gnotobiotic system has been developed to study the interaction between Medicago truncatula Jemalong J5 and the mutualistic bacteria Pseudomonas fluorescens strain C7R12 under two atmospheric CO₂ concentrations: ambient (365 ppm) versus enriched (750 ppm). Costs and benefits for each partner have been determined over time by measuring plant development and growth, the C and N contents of the various plant parts and the density of the bacteria in rhizosphere compartments. Following the increase in CO₂, there was a beneficial effect of P. fluorescens C7R12 on development, vegetative growth, and C/N content of M. truncatula. Concerning plant reproduction, an early seed production was noticed in presence of the bacterial strain combined with increased atmospheric CO₂ conditions. Paradoxically, this transient increase in seed production was correlated with a decrease in bacterial density in the rhizosphere soil, revealing a cost of increased CO₂ for the bacterial strain. This shift of costs-benefits ratio disappeared later during the plant growth. In conclusion, the increase in CO₂ concentration modifies transiently the cost-benefit balance in favor of the plant. These results may be explained either by a competition between the two partners or a change in bacterial physiology. The ecosystem functioning depends on the stability of many plant-microbe associations that abiotic factors can disrupt.     

Natural Transformation of Acinetobacter sp. Strain BD413 with Cell Lysates of Acinetobacter sp., Pseudomonas fluorescens, and Burkholderia cepacia in Soil Microcosms

To elucidate the biological significance of dead bacterial cells in soil to the intra- and interspecies transfer of gene fragments by natural transformation, we have exposed the kanamycin-sensitive recipient Acinetobacter sp. strain BD413(pFG4) to lysates of the kanamycin-resistant donor bacteria Acinetobacter spp., Pseudomonas fluorescens, and Burkholderia cepacia. Detection of gene transfer was facilitated by the recombinational repair of a partially (317 bp) deleted kanamycin resistance gene in the recipient bacterium. The investigation revealed a significant potential of these DNA sources to transform Acinetobacter spp. residing both in sterile and in nonsterile silt loam soil. Heat-treated (80°C, 15 min) cell lysates were capable of transforming strain BD413 after 4 days of incubation in sterile soil and for up to 8 h in nonsterile soil. Transformation efficiencies obtained in vitro and in situ with the various lysates were similar to or exceeded those obtained with conventionally purified DNA. The presence of cell debris did not inhibit transformation in soil, and the debris may protect DNA from rapid biological inactivation. Natural transformation thus provides Acinetobacter spp. with an efficient mechanism to access genetic information from different bacterial species in soil. The relatively short-term biological activity (e.g., transforming activity) of chromosomal DNA in soil contrasts the earlier reported long-term physical stability of DNA, where fractions have been found to persist for several weeks in soil. Thus, there seems to be a clear difference between the physical and the functional significance of chromosomal DNA in soil.     

Natural transformation of Acinetobacter sp. strain BD413 with cell lysates of Acinetobacter sp., Pseudomonas fluorescens, and Burkholderia cepacia in soil microcosms

To elucidate the biological significance of dead bacterial cells in soil to the intra- and interspecies transfer of gene fragments by natural transformation, we have exposed the kanamycin-sensitive recipient Acinetobacter sp. strain BD413(pFG4) to lysates of the kanamycin-resistant donor bacteria Acinetobacter spp., Pseudomonas fluorescens, and Burkholderia cepacia. Detection of gene transfer was facilitated by the recombinational repair of a partially (317 bp) deleted kanamycin resistance gene in the recipient bacterium. The investigation revealed a significant potential of these DNA sources to transform Acinetobacter spp. residing both in sterile and in nonsterile silt loam soil. Heat-treated (80 degrees C, 15 min) cell lysates were capable of transforming strain BD413 after 4 days of incubation in sterile soil and for up to 8 h in nonsterile soil. Transformation efficiencies obtained in vitro and in situ with the various lysates were similar to or exceeded those obtained with conventionally purified DNA. The presence of cell debris did not inhibit transformation in soil, and the debris may protect DNA from rapid biological inactivation. Natural transformation thus provides Acinetobacter spp. with an efficient mechanism to access genetic information from different bacterial species in soil. The relatively short-term biological activity (e.g., transforming activity) of chromosomal DNA in soil contrasts the earlier reported long-term physical stability of DNA, where fractions have been found to persist for several weeks in soil. Thus, there seems to be a clear difference between the physical and the functional significance of chromosomal DNA in soil.     

In Silico Analysis of Usher Encoding Genes in Klebsiella pneumoniae and Characterization of Their Role in Adhesion and Colonization

Chaperone/usher (CU) assembly pathway is used by a wide range of Enterobacteriaceae to assemble adhesive surface structures called pili or fimbriae that play a role in bacteria-host cell interactions. In silico analysis revealed that the genome of Klebsiella pneumoniae LM21 harbors eight chromosomal CU loci belonging to γκп and ϭ clusters. Of these, only two correspond to previously described operons, namely type 1 and type 3-encoding operons. Isogenic usher deletion mutants of K. pneumoniae LM21 were constructed for each locus and their role in adhesion to animal (Intestine 407) and plant (Arabidopsis thaliana) cells, biofilm formation and murine intestinal colonization was investigated. Type 3 pili usher deleted mutant was impaired in all assays, whereas type 1 pili usher deleted mutant only showed attenuation in adhesion to plant cells and in intestinal colonization. The LM21ΔkpjC mutant was impaired in its capacity to adhere to Arabidopsis cells and to colonize the murine intestine, either alone or in co-inoculation experiments. Deletion of LM21kpgC induced a significant decrease in biofilm formation, in adhesion to animal cells and in colonization of the mice intestine. The LM21∆kpaC and LM21∆kpeC mutants were only attenuated in biofilm formation and the adhesion abilities to Arabidopsis cells, respectively. No clear in vitro or in vivo effect was observed for LM21∆kpbC and LM21∆kpdC mutants. The multiplicity of CU loci in K. pneumoniae genome and their specific adhesion pattern probably reflect the ability of the bacteria to adhere to different substrates in its diverse ecological niches.     

Aspergillus felis sp. nov., an Emerging Agent of Invasive Aspergillosis in Humans,Cats, and Dogs

We describe a novel heterothallic species in Aspergillus section Fumigati, namely A. felis (neosartorya-morph) isolated from three host species with invasive aspergillosis including a human patient with chronic invasive pulmonary aspergillosis, domestic cats with invasive fungal rhinosinusitis and a dog with disseminated invasive aspergillosis. Disease in all host species was often refractory to aggressive antifungal therapeutic regimens. Four other human isolates previously reported as A. viridinutans were identified as A. felis on comparative sequence analysis of the partial β-tubulin and/or calmodulin genes. A. felis is a heterothallic mold with a fully functioning reproductive cycle, as confirmed by mating-type analysis, induction of teleomorphs within 7 to 10 days in vitro and ascospore germination. Phenotypic analyses show that A. felis can be distinguished from the related species A. viridinutans by its ability to grow at 45°C and from A. fumigatus by its inability to grow at 50°C. Itraconazole and voriconazole cross-resistance was common in vitro.     

Streptococcus pneumoniae GAPDH Is Released by Cell Lysis and Interacts with Peptidoglycan

Release of conserved cytoplasmic proteins is widely spread among Gram-positive and Gram-negative bacteria. Because these proteins display additional functions when located at the bacterial surface, they have been qualified as moonlighting proteins. The GAPDH is a glycolytic enzyme which plays an important role in the virulence processes of pathogenic microorganisms like bacterial invasion and host immune system modulation. However, GAPDH, like other moonlighting proteins, cannot be secreted through active secretion systems since they do not contain an N-terminal predicted signal peptide. In this work, we investigated the mechanism of GAPDH export and surface retention in Streptococcus pneumoniae, a major human pathogen. We addressed the role of the major autolysin LytA in the delivery process of GAPDH to the cell surface. Pneumococcal lysis is abolished in the ΔlytA mutant strain or when 1% choline chloride is added in the culture media. We showed that these conditions induce a marked reduction in the amount of surface-associated GAPDH. These data suggest that the presence of GAPDH at the surface of pneumococcal cells depends on the LytA-mediated lysis of a fraction of the cell population. Moreover, we demonstrated that pneumococcal GAPDH binds to the bacterial cell wall independently of the presence of the teichoic acids component, supporting peptidoglycan as a ligand to surface GAPDH. Finally, we showed that peptidoglycan-associated GAPDH recruits C1q from human serum but does not activate the complement pathway.